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  • Different degradation rates of nanofiber vascular grafts in small and large animal models
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2020-01-22
    Takuma Fukunishi; Chin Siang Ong; Pooja Yesantharao; Cameron A. Best; Tai Yi; Huaitao Zhang; Gunnar Mattson; Joseph Boktor; Kevin Nelson; Toshiharu Shinoka; Christopher K. Breuer; Jed Johnson; Narutoshi Hibino

    Nanofiber vascular grafts have been shown to create neovessels made of autologous tissue, by in vivo scaffold biodegradation over time. However, many studies on graft materials and biodegradation have been conducted in vitro or in small animal models, instead of large animal models, which demonstrate different degradation profiles. In this study, we compared the degradation profiles of nanofiber vascular grafts in a rat model and a sheep model, while controlling for the type of graft material, the duration of implantation, fabrication method, type of circulation (arterial/venous), and type of surgery (interposition graft). We found that there was significantly less remaining scaffold (i.e., faster degradation) in nanofiber vascular grafts implanted in the sheep model compared with the rat model, in both the arterial and the venous circulations, at 6 months postimplantation. In addition, there was more extracellular matrix deposition, more elastin formation, more mature collagen, and no calcification in the sheep model compared with the rat model. In conclusion, studies comparing degradation of vascular grafts in large and small animal models remain limited. For clinical translation of nanofiber vascular grafts, it is important to understand these differences.

  • Engineered skin graft with stromal vascular fraction cells encapsulated in fibrin–collagen hydrogel: A clinical study for diabetic wound healing
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2020-01-17
    Mohammad Ali Nilforoushzadeh; Mahsa Mollapour Sisakht; Mohammad Amir Amirkhani; Alexander M. Seifalian; Hamid Reza Banafshe; Javad Verdi; Mehdi Nouradini

    Despite the abundance of skin substitutes in the worldwide market, major hurdles in developing more complex tissues include the addition of skin appendages and vascular networks as the most important structure. The aim of this research was a clinical feasibility study of a novel prevascularized skin grafts containing the dermal and epidermal layer using the adipose stromal vascular fraction (SVF)‐derived endothelial cell population for vascular network regeneration. Herein, we characterized hydrogel with emphasis on biological compatibility and cell proliferation, migration, and vitality. The therapeutic potential of the prevascularized hydrogel transplanted on five human subjects as an intervention group with diabetic wounds was compared with nonvascularized skin grafts as the control on five patients. Wound planimetric and biometric analysis was performed using a Mann–Whitney nonparametric t‐test (p ≤ .05). The fibrin–collagen hydrogel was suitable for skin organotypic cell culture. There was a significant (p ≤ .05) increased in skin thickness and density in the vascular beds of the hypodermis measured with skin scanner compared with that in the control group. No significant macroscopic differences were observed between the intervention and control groups (p ≤ .05). In summary, we report for the first time the use of autologous dermal–epidermal skin grafts with intrinsic vascular plexus in a clinical feasibility study. The preliminary data showed that SVF‐based full‐thickness skin grafts are safe and accelerate the wound healing process. The next stage of the study is a full‐scale randomized clinical trial for the treatment of patients with chronic wounds.

  • The role of microbiota in tissue repair and regeneration
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2020-01-15
    Amin Shavandi; Pouya Saeedi; Philippe Gérard; Esmat Jalalvandi; David Cannella; Alaa El‐Din Bekhit

    A comprehensive understanding of the human body endogenous microbiota is essential for acquiring an insight into the involvement of microbiota in tissue healing and regeneration process in order to enable development of biomaterials with a better integration with human body environment. Biomaterials used for biomedical applications are normally germ‐free, and the human body as the host of the biomaterials is not germ‐free. The complexity and role of the body microbiota in tissue healing/regeneration have been underestimated historically. Traditionally, studies aiming at the development of novel biomaterials had focused on the effects of environment within the target tissue, neglecting the signals generated from the microbiota and their impact on tissue regeneration. The significance of the human body microbiota in relation to metabolism, immune system, and consequently tissue regeneration has been recently realised and is a growing research field. This review summarises recent findings on the role of microbiota and mechanisms involved in tissue healing and regeneration, in particular skin, liver, bone, and nervous system regrowth and regeneration highlighting the potential new roles of microbiota for development of a new generation of biomaterials.

  • Co‐transplantation of Wharton's jelly mesenchymal stem cell‐derived osteoblasts with differentiated endothelial cells does not stimulate blood vessel and osteoid formation in nude mice models
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2020-01-10
    Marie Naudot; Anaïs Barre; Alexandre Caula; Henri Sevestre; Stéphanie Dakpé; Andreas Albert Mueller; Bernard Devauchelle; Sylvie Testelin; Jean Pierre Marolleau; Sophie Le Ricousse

    A major challenge in bone tissue engineering is the lack of post‐implantation vascular growth into biomaterials. In the skeletal system, blood vessel growth appears to be coupled to osteogenesis—suggesting the existence of molecular crosstalk between endothelial cells (ECs) and osteoblastic cells. The present study (performed in two murine ectopic models) was designed to determine whether co‐transplantation of human Wharton's jelly mesenchymal stem cell‐derived osteoblasts (WJMSC‐OBs) and human differentiated ECs enhances bone regeneration and stimulates angiogenesis, relative to the seeding of WJMSC‐OBs alone.

  • β‐Adrenergic stimuli and rotating suspension culture enhance conversion of human adipogenic mesenchymal stem cells into highly conductive cardiac progenitors
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2020-01-10
    Jose Francisco Islas; Reza Abbasgholizadeh; Clifford Dacso; Vladimir N. Potaman; Stephen Navran; Richard A. Bond; Dinakar Iyer; Ravi Birla; Robert J. Schwartz

    Clinical trials using human adipogenic mesenchymal stem cells (hAdMSCs) for the treatment of cardiac diseases have shown improvement in cardiac function and were proven safe. However, hAdMSCs do not convert efficiently into cardiomyocytes (CMs) or vasculature. Thus, reprogramming hAdMSCs into myocyte progenitors may fare better in future investigations. To reprogramme hAdMSCs into electrically conductive cardiac progenitor cells, we pioneered a three‐step reprogramming strategy that uses proven MESP1/ETS2 transcription factors, β‐adrenergic and hypoxic signalling induced in three‐dimensional (3D) cardiospheres. In Stage 1, ETS2 and MESP1 activated NNKX2.5, TBX5, MEF2C, dHAND, and GATA4 during the conversion of hAdMSCs into cardiac progenitor cells. Next, in Stage 2, β2AR activation repositioned cardiac progenitors into de novo immature conductive cardiac cells, along with the appearance of RYR2, CAV2.1, CAV3.1, NAV1.5, SERCA2, and CX45 gene transcripts and displayed action potentials. In Stage 3, electrical conduction that was fostered by 3D cardiospheres formed in a Synthecon®, Inc. rotating bioreactor induced the appearance of hypoxic genes: HIF‐1α/β, PCG 1α/β, and NOS2, which coincided with the robust activation of adult contractile genes including MLC2v, TNNT2, and TNNI3, ion channel genes, and the appearance of hyperpolarization‐activated and cyclic nucleotide‐gated channels (HCN1–4). Conduction velocities doubled to ~200 mm/s after hypoxia and doubled yet again after dissociation of the 3D cell clusters to ~400 mm/s. By comparison, normal conduction velocities within working ventricular myocytes in the whole heart range from 0.5 to 1 m/s. Epinephrine stimulation of stage 3 cardiac cells in patches resulted in an increase in amplitude of the electrical wave, indicative of conductive cardiac cells. Our efficient protocol that converted hAdMSCs into highly conductive cardiac progenitors demonstrated the potential utilization of stage 3 cells for tissue engineering applications for cardiac repair.

  • Interaction of material stiffness and negative pressure to enhance differentiation of bone marrow‐derived stem cells and osteoblast proliferation
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2020-01-09
    Rui Wang; Patrick Thayer; Aaron Goldstein; William D. Wagner

    Negative pressure wound therapy (NPWT) results in improved wound repair and the combined use of NPWT with elastomeric materials may further stimulate and accelerate tissue repair. No firmly established treatment modalities using both NPWT and biomaterials exist for orthopedic application. The goal of this study was to investigate the response of osteoblasts and bone marrow‐derived mesenchymal stem cells to negative pressure and to determine whether a newly developed elastic osteomimetic bone repair material (BRM), a blend of type I collagen, chondroitin 6‐sulfate, and poly (octanediol citrate) could enhance the osteoblastic phenotype. The results indicate that proliferation and alkaline phosphatase activity of hFOB1.19 osteoblasts were significantly increased with exposure to 12 hr of negative pressure (−125 mmHg). Follow‐on studies with rat and human mesenchymal stem cells confirmed that negative pressure enhanced osteoblastic maturation. In addition, a significant interaction of negative pressure and electrospun BRM resulted in increased mRNA expression of alkaline phosphatase, osteopontin, collagen1α2, and HIF1α, whereas little or no effect on these genes was observed on electrospun collagen or tissue culture plastic. Together, these results suggest that the use of this novel biomaterial, BRM, with NPWT may ultimately translate into a safe and cost‐effective clinical application to accelerate bone repair.

  • Development of a protocol for maintaining viability while shipping organoid‐derived retinal tissue
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2020-01-06
    Ratnesh K. Singh; Paige Winkler; Francois Binette; Randolph D. Glickman; Magdalene Seiler; Simon M. Petersen‐Jones; Igor O. Nasonkin

    Retinal organoid technology enables generation of an inexhaustible supply of three‐dimensional retinal tissue from human pluripotent stem cells (hPSCs) for regenerative medicine applications. The high similarity of organoid‐derived retinal tissue and transplantable human fetal retina provides an opportunity for evaluating and modeling retinal tissue replacement strategies in relevant animal models in the effort to develop a functional retinal patch to restore vision in patients with profound blindness caused by retinal degeneration. Because of the complexity of this very promising approach requiring specialized stem cell and grafting techniques, the tasks of retinal tissue derivation and transplantation are frequently split between geographically distant teams. Delivery of delicate and perishable neural tissue such as retina to the surgical sites requires a reliable shipping protocol and also controlled temperature conditions with damage‐reporting mechanisms in place to prevent transplantation of tissue damaged in transit into expensive animal models. We have developed a robust overnight tissue shipping protocol providing reliable temperature control, live monitoring of the shipment conditions and physical location of the package, and damage reporting at the time of delivery. This allows for shipping of viable (transplantation‐competent) hPSC‐derived retinal tissue over large distances, thus enabling stem cell and surgical teams from different parts of the country to work together and maximize successful engraftment of organoid‐derived retinal tissue. Although this protocol was developed for preclinical in vivo studies in animal models, it is potentially translatable for clinical transplantation in the future and will contribute to developing clinical protocols for restoring vision in patients with retinal degeneration.

  • Evaluation of keratin biomaterial containing silver nanoparticles as a potential wound dressing in full‐thickness skin wound model in diabetic mice
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2020-01-03
    Marek Konop; Joanna Czuwara; Ewa Kłodzińska; Anna K. Laskowska; Dorota Sulejczak; Tatsiana Damps; Urszula Zielenkiewicz; Iwona Brzozowska; Antonio Sureda; Tomasz Kowalkowski; Robert A. Schwartz; Lidia Rudnicka

    Keratin is a cytoskeletal scaffolding protein essential for wound healing and tissue recovery. The aim of the study was to evaluate the potential role of insoluble fur keratin‐derived powder containing silver nanoparticles (FKDP‐AgNP) in the allogenic full‐thickness surgical skin wound model in diabetic mice. The scanning electron microscopy image evidenced that the keratin surface is covered by a single layer of silver nanoparticles. Data obtained from dynamic light scattering and micellar electrokinetic chromatography showed three fractions of silver nanoparticles with an average diameter of 130, 22.5, and 5 nm. Microbiologic results revealed that the designed insoluble FKDP‐AgNP dressing to some extent inhibit the growth of Escherichia coli and Staphylococcus aureus. In vitro assays showed that the FKDP‐AgNP dressing did not inhibit fibroblast growth or induce hemolysis. In vivo studies using a diabetic mice model confirmed biocompatible properties of the insoluble keratin dressings. FKDP‐AgNP significantly accelerated wound closure and epithelization at Days 5 and 8 (p < .05) when compared with controls. Histological examination of the inflammatory response documented that FKDP‐AgNP‐treated wounds contained predominantly macrophages, whereas their untreated variants showed mixed cell infiltrates rich in neutrophils. Wound inflammatory response based on macrophages favors tissue remodeling and healing. In conclusion, the investigated FKDP‐AgNP dressing consisting of an insoluble fraction of keratin, which is biocompatible, significantly accelerated wound healing in a diabetic mouse model.

  • Design of a new dual mesh with an absorbable nanofiber layer as a potential implant for abdominal hernia treatment
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2019-12-30
    Mehmet Kaya; Zehra Betul Ahi; Emre Ergene; Pinar Yilgor Huri; Kadriye Tuzlakoglu

    Dual meshes are often preferred in the treatment of umbilical and incisional hernias where the abdominal wall defect is large. These meshes are generally composed of either two nonabsorbable layers or a nonabsorbable layer combined with an absorbable one that degrades within the body upon healing of the defect. The most crucial point in the design of a dual mesh is to produce the respective layers based on the structure and requirements of the recipient site. We herein developed a dual mesh that consists of two layers: a nanofibrous layer made of poly (glycerol sebacate)/poly (caprolactone) (PGS/PCL) to support the healing of the abdominal wall defect and a nondegradable, nonadhesive smooth layer made of polycarbonateurethane (PU) with suitable properties to avoid the adhesion of the viscera to the mesh. To prepare the double‐sided structure, PGS/PCL was directly electrospun onto the PU film. This processing approach provided a final product with well‐integrated layers as observed by a scanning electron microscope. Tensile test performed at the dry state of the samples showed that the dual mesh has the ability to elongate seven times more as compared with the commercially available counterparts, mimicking the native tissue properties. The degradation test carried out at physiological conditions revealed that PGS started to degrade within the first 15 days. in vitro studies with human umbilical vein endothelial cells demonstrated the double function of the meshes, in which PU layer did not allow cell adhesion, whereas PGS/PCL layer has the ability to support cell adhesion and proliferation. Therefore, the material developed in this study has the potential to be an alternative to the existing hernia mesh products.

  • Wound healing grafts: Omega‐3 fatty acid lipid content differentiates the lipid profiles of acellular Atlantic cod skin from traditional dermal substitutes
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2019-12-30
    Aristotelis Kotronoulas; Hulda S. Jónasdóttir; Rósa S. Sigurðardóttir; Skarphéðinn Halldórsson; Guðmundur G. Haraldsson; Óttar Rolfsson

    Acellular fish skin (ACS) has emerged as a dermal substitute used to promote wound healing with decreased scar formation and pain relief that may be due to polyunsaturated fatty acid (PUFA) content. However, the PUFA content of ACS is still unknown. The aim of this study was to compare the total fatty acids and lipid profiles of ACS to two bovine‐based grafts and standard of care human cadaver skin (HCS). Furthermore, there was also the goal to assess the capability of ACS lipid content to enhance wound healing. The fatty acid analysis was performed with GC–FID, and an LC–MS untargeted method was developed in order to the analyse the lipid profiles of the grafts was. The enhancement of wound healing by the ACS extract was investigated in vitro on HaCat cells. Our results showed that ACS had the highest content of PUFA (27.0 ± 1.43% of their total fatty acids), followed by HCS (20.6 ± 3.9%). The two grafts of bovine origin presented insignificant PUFA amounts. The majority of the PUFAs found in ACS were omega‐3, and in HCS, they were omega‐6. The untargeted lipidomics analysis demonstrated that ACS grafts were characterized by phosphatidylcholine containing either 20:5 or 22:6 omega‐3 PUFA. The ACS lipid extract increased the HaCat cells migration and enhanced wound closure 4 hr earlier versus control. Our study demonstrated that ACS has a lipid profile that is distinct from other wound healing grafts, that PUFAs are maintained in ACS post‐processing as phosphatidylcholine, and that ACS lipid content influences wound healing properties.

  • Tissue engineering and regenerative medicine strategies for the female breast
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2019-12-30
    Claudio Conci; Lorenzo Bennati; Chiara Bregoli; Federica Buccino; Francesca Danielli; Michela Gallan; Ereza Gjini; Manuela T. Raimondi

    The complexity of mammary tissue and the variety of cells involved make tissue regeneration an ambitious goal. This review, supported by both detailed macro and micro anatomy, illustrates the potential of regenerative medicine in terms of mammary gland reconstruction to restore breast physiology and morphology, damaged by mastectomy. Despite the widespread use of conventional therapies, many critical issues have been solved using the potential of stem cells resident in adipose tissue, leading to commercial products. in vitro research has reported that adipose stem cells are the principal cellular source for reconstructing adipose tissue, ductal epithelium, and nipple structures. In addition to simple cell injection, construct made by cells seeded on a suitable biodegradable scaffold is a viable alternative from a long‐term perspective. Preclinical studies on mice and clinical studies, most of which have reached Phase II, are essential in the commercialization of cellular therapy products. Recent studies have revealed that the enrichment of fat grafting with stromal vascular fraction cells is a viable alternative to breast reconstruction. Although in the future, organ‐on‐a‐chip can be envisioned, for the moment researchers are still focusing on therapies that are a long way from regenerating the whole organ, but which nevertheless prevent complications, such as relapse and loss in terms of morphology.

  • Design, construction, and biological testing of an implantable porous trilayer scaffold for repairing osteoarthritic cartilage
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2019-12-26
    Yaima Campos; Francisco J. Sola; Amisel Almirall; Gastón Fuentes; Christina Eich; Ivo Que; Alan Chan; Eric Kaijzel; Yasuhiko Tabata; Luis Quintanilla; José C. Rodríguez‐Cabello; Luis J. Cruz

    Various tissue engineering systems for cartilage repair have been designed and tested over the past two decades, leading to the development of many promising cartilage grafts. However, no one has yet succeeded in devising an optimal system to restore damaged articular cartilage. Here, the design, assembly, and biological testing of a porous, chitosan/collagen‐based scaffold as an implant to repair damaged articular cartilage is reported. Its gradient composition and trilayer structure mimic variations in natural cartilage tissue. One of its layers includes hydroxyapatite, a bioactive component that facilitates the integration of growing tissue on local bone in the target area after scaffold implantation. The scaffold was evaluated for surface morphology; rheological performance (storage, loss, complex, and time‐relaxation moduli at 1 kHz); physiological stability; in vitro activity and cytotoxicity (on a human chondrocyte C28 cell line); and in vivo performance (tissue growth and biodegradability), in a murine model of osteoarthritis. The scaffold was shown to be mechanically resistant and noncytotoxic, favored tissue growth in vivo, and remained stable for 35 days postimplantation in mice. These encouraging results highlight the potential of this porous chitosan/collagen scaffold for clinical applications in cartilage tissue engineering.

  • Efficacy of human HC016 cell transplants on neuroprotection and functional recovery in a rat model of acute spinal cord injury
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2019-12-26
    Alfredo Maqueda; Francisco J. Rodriguez

    Spinal cord injury (SCI) is a devastating event with huge personal and social costs, for which there is no effective treatment. Cell therapy constitutes a promising therapeutic approach for SCI; however, its clinical potential is seriously limited by their low survival in the hostile conditions encompassing the acute phase of SCI. Human HC016 (hHC016) cells, generated from expanded human adipose mesenchymal stem cells (hAMSCs) and pulsed with a patented protocol with hydrogen peroxide (H2O2), are expected to acquire improved resistance to oxidative environments which appears as a major limiting factor hampering the engrafting success. Our specific aim was to assess whether H2O2‐pulsed hHC016 cells had an improved survival and thus therapeutic efficacy in a rat contusion model of acute SCI when grafted 48 hr after injury. Functional recovery was evaluated up to 56 days post‐injury (dpi) by locomotor (open field test and CatWalk) and sensory (Von Frey and Hargreaves) tests. Besides, histological evaluation of transplanted cell survival and tissue protection/regeneration was also performed. Functional results showed a statistically significant improvement on locomotor recovery outcomes with hHC016 cells. Accordingly, superior cell survival in correlation with long‐term neuroprotection, higher axonal regeneration, and reduced astroglial and microglial reactivity was also observed with hHC016 cells. These results demonstrate an enhanced survival capacity of hHC016 cells resulting in improved functional and histological outcomes as compared with hAMSCs, indicating that hHC016 cell transplants may constitute a promising cell therapy for acute SCI.

  • Intratendon delivery of leukocyte‐rich platelet‐rich plasma at early stage promotes tendon repair in a rabbit Achilles tendinopathy model
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2019-12-23
    Sihao Li; Yifan Wu; Guangyao Jiang; Xiulian Tian; Jianqiao Hong; Shiming Chen; Ruijian Yan; Gang Feng; Zhiyuan Cheng

    Tendinopathy is a great obstacle in clinical practice due to its poor regenerative capacity. The influence of different stages of tendinopathy on effects of leukocyte‐rich platelet‐rich plasma (Lr‐PRP) has not been elucidated. The aim of this study is to investigate the optimal time point for delivery of Lr‐PRP on tendinopathy. A tendinopathy model was established by local collagenase injection on the rabbit Achilles tendon. Then after collagenase induction, following treatments were applied randomly on the lesion: (a) 200 μl of Lr‐PRP at 1 week (PRP‐1 group), (b) 200 μl of saline at 1 week (Saline‐1 group), (c) 200 μl of Lr‐PRP at 4 weeks (PRP‐2 group), and (d) 200 μl of saline at 4 weeks (Saline‐2 group). Six weeks after collagenase induction, outcomes were assessed by magnetic resonance imaging, cytokine quantification, gene expression, histology, and transmission electron microscopy. Our results demonstrated that PRP‐1 group had the least cross‐sectional area and lesion percent of the involved tendon, as well as the lowest signal intensity in magnetic resonance imaging among all groups. However, the PRP‐2 group showed larger cross‐sectional area than saline groups. Enzyme‐linked immunosorbent assay indicated that PRP‐1 group had a higher level of interleukin‐10 but lower level of interleukin‐6 when compared with PRP‐2 and saline groups. Meanwhile, the highest expression of collagen (Col) 1 in PRP‐1 and Col 3, matrix metalloproteinase (MMP)‐1, and MMP‐3 in PRP‐2 was found. Histologically, the PRP‐1 showed better general scores than PRP‐2, and no significant difference was found between the PRP‐2 and saline groups. For transmission electron microscopy, PRP‐1 had the largest mean collagen fibril diameter, and the PRP‐2 group showed even smaller mean collagen fibril diameter than saline groups. In conclusion, intratendon delivery of Lr‐PRP at early stage showed beneficial effect for repair of tendinopathy but not at late stage. For translation of our results to clinical circumstances, further studies are still needed.

  • Astrocytes‐derived exosomes induce neuronal recovery after traumatic brain injury via delivering gap junction alpha 1‐20 k
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2019-12-22
    Wei Chen; Ping Zheng; Tao Hong; Yang Wang; Ning Liu; Bin He; Shufeng Zou; Dabin Ren; Jian Duan; Lin Zhao; Jiugeng Feng

    Astrocytes are more resistant to ischemia and hypoxia in the acute phase of brain injury after traumatic brain injury (TBI). Previous study showed that gap junction alpha 1 (GJA1) phosphorylation can increase the survival of damaged astrocytes. The GJA1‐20 k expression in neurons co‐culture with astrocytes was positively correlated with exosomes uptake. This study aims to explore the effect of exogenous GJA1‐20 k carried by astrocyte‐derived exosomes on neurons apoptosis and mitochondrial function after TBI. Astrocytes were co‐cultured with the neuron with/without damage from air pressure. Exosomes were isolated, extracted from the culture medium by differential ultra‐centrifugation, and verified by electron microscopy. Immunofluorescence staining, tunnel, western blot were employed to detect exosomes marker CD60, apoptosis, and mitochondrial function related protein expression and GJA1‐20 k in cell culture. A rat model of hydraulic injury TBI was built, and exosomes was transferred. 2,3,5‐Triphenyltetrazolium chloride (TTC) staining and immunohistochemistry staining of Nissl and microtubule associated protein 2 were used to detect the brain damage. A transwell stereo culture model of astrocytes and TBI‐like injured neuron was constructed. The exosomes derived from astrocytes promoted the recovery of damaged neuron by in vitro exosome treatment. Compared with GJA1‐20 k knockout exosome control group, GJA1‐20 k exosomes were uptaken by neuron and downregulated the apoptosis rate and upregulated mitochondrial function to promote neuronal recovery. Finally, the results were validated by TTC staining and damaged tissue sections of rat TBI model. This study contributes to a better understanding of the astrocyte‐neuron protection mechanism in TBI and provides a potential new target for the treatment of TBI.

  • Limitations of recellularized biological scaffolds for human transplantation
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2019-12-22
    Claudia Bilodeau; Olivia Goltsis; Ian M. Rogers; Martin Post

    A shortage of donor organs for transplantation and the dependence of the recipients on immunosuppressive therapy have motivated researchers to consider alternative regenerative approaches. The answer may reside in acellular scaffolds generated from cadaveric human and animal tissues. Acellular scaffolds are expected to preserve the architectural and mechanical properties of the original organ, permitting cell attachment, growth, and differentiation. Although theoretically, the use of acellular scaffolds for transplantation should pose no threat to the recipient's immune system, experimental data have revealed significant immune responses to allogeneic and xenogeneic transplanted scaffolds. Herein, we review the various factors of the scaffold that could trigger an inflammatory and/or immune response, thereby compromising its use for human transplant therapy. In addition, we provide an overview of the major cell types that have been considered for recellularization of the scaffold and their potential contribution to triggering an immune response.

  • Effect of heart ischemia and administration route on biodistribution and transduction efficiency of AAV9 vectors
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2019-12-22
    Paula García‐Olloqui; Juan Roberto Rodriguez‐Madoz; Marianna Di Scala; Gloria Abizanda; África Vales; Cristina Olagüe; Olalla Iglesias‐García; Eduardo Larequi; Laura Pilar Aguado‐Alvaro; Adrián Ruiz‐Villalba; Felipe Prosper; Gloria Gonzalez‐Aseguinolaza; Beatriz Pelacho

    Adeno‐associated viruses (AAV) have become one of the most promising tools for gene transfer in clinics. Among all the serotypes, AAV9 has been described as the most efficient for cardiac transduction. In order to achieve optimal therapeutic delivery in heart disease, we have explored AAV9 transduction efficiency in an infarcted heart using different routes of administration and promoters, including a cardiac‐specific one. AAV9 vectors carrying luciferase or green fluorescence protein under the control of the ubiquitous elongation‐factor‐1‐alpha or the cardiac‐specific troponin‐T (TnT) promoters were administered by intramyocardial or intravenous injection, either in healthy or myocardial‐infarcted mice. The transduction efficacy and specificity, the time‐course expression, and the safety of each vector were tested. High transgene expression levels were found in the heart, but not in the liver, of mice receiving AAV‐TnT, which was significantly higher after intramyocardial injection regardless of ischemia‐induction. On the contrary, high hepatic transgene expression levels were detected with the elongation‐factor‐1‐alpha‐promoter, independently of the administration route and heart damage. Moreover, tissue‐specific green fluorescence protein expression was found in cardiomyocytes with the TnT vector, whereas minimal cardiac expression was detected with the ubiquitous one. Interestingly, we found that myocardial infarction greatly increased the transcriptional activity of AAV genomes. Our findings show that the use of cardiac promoters allows for specific and stable cardiac gene expression, which is optimal and robust when intramyocardially injected. Furthermore, our data indicate that the pathological status of the tissue can alter the transcriptional activity of AAV genomes, an aspect that should be carefully evaluated for clinical applications.

  • Mineralized nanofibrous scaffold promotes phenamil‐induced osteoblastic differentiation while mitigating adipogenic differentiation
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2019-12-21
    Yangxi Liu; Jue Hu; Hongli Sun

    Large bone defects represent a significant unmet medical challenge. Cost effectiveness and better stability make small molecule organic compounds a more promising alternative compared with biomacromolecules, for example, growth factors/hormones, in regenerative medicine. However, one common challenge for the application of these small compounds is their side‐effect issue. Phenamil is emerging as an intriguing small molecule to promote bone repair by strongly activating bone morphogenetic protein signaling pathway. In addition to osteogenesis, phenamil also induces significant adipogenesis based on some in vitro studies, which is a concern that impedes it from potential clinical applications. Besides the soluble chemical signals, cellular differentiation is heavily dependent on the microenvironments provided by the 3D scaffolds. Therefore, we developed a 3D nanofibrous biomimetic scaffold‐based strategy to harness the phenamil‐induced stem cell lineage differentiation. Based on the gene expression, alkaline phosphatase activity, and mineralization data, we indicated that bone‐matrix mimicking mineralized‐gelatin nanofibrous scaffold effectively improved phenamil‐induced osteoblastic differentiation, while mitigating the adipogenic differentiation in vitro. In addition to normal culture conditions, we also indicated that mineralized matrix can significantly improve phenamil‐induced osteoblastic differentiation in simulated inflammatory condition. In viewing of the crucial role of mineralized matrix, we developed an innovative and facile mineral deposition‐based strategy to sustain release of phenamil from 3D scaffolds for efficient local bone regeneration. Overall, our study demonstrated that biomaterials played a crucial role in modulating small molecule drug phenamil‐induced osteoblastic differentiation by providing a bone‐matrix mimicking mineralized gelatin nanofibrous scaffolds.

  • Specific complexes derived from extracellular matrix facilitate generation of structural and drug‐responsive human salivary gland microtissues through maintenance stem cell homeostasis
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2019-12-19
    Siqi Zhang; Yi Sui; Xiaoming Fu; Yanrui Feng; Zuyuan Luo; Yuanyuan Zhang; Shicheng Wei

    Three‐dimensional cultured salivary glands (SGs) microtissues hold great potentials for clinical research. However, most SGs microtissues still lack convincing structure and function due to poor supplementation of factors to maintain stem cell homeostasis. Extracellular matrix (ECM) plays a crucial role in regulating stem cell behavior. Thus, it is necessary to model stem cell microenvironment in vitro by supplementing culture medium with proteins derived from ECM. We prepared specific complexes from human SG ECM (s‐Ecx) and analyzed the components of the s‐Ecx. Human SG epithelial and mesenchymal cells were used to generate microtissues, and the optimum seeding cell number and ratio of two cell types were determined. Then, the s‐Ecx was introduced to the culture medium to assess its effect on stem cell behavior. Multiple specific factors were presented in s‐Ecx. s‐Ecx promoted maintenance of the stem cell and formation of specific structures resembling that of salivary glands and containing mucins, which suggested stem cell differentiation potential. Moreover, treatment of the microtissues with s‐Ecx increased their sensitivity to neurotransmitters. On the basis of the analysis of components, we believed that the presented growth factors are able to interact with stem cell they encountered in vivo, which promote the capacity to maintain stem cell homeostasis. This work provided foundations to study molecular mechanism of stem cell homeostasis in SGs and develop novel therapies for dry mouth through new drug discovery and disease modeling.

  • Synthesis and characterization of osteoinductive visible light‐activated adhesive composites with antimicrobial properties
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2019-12-18
    Amirhossein Moghanian; Roberto Portillo‐Lara; Ehsan Shirzaei Sani; Hailey Konisky; Seyed Hossein Bassir; Nasim Annabi

    Orthopedic surgical procedures based on the use of conventional biological graft tissues are often associated with serious post‐operative complications such as immune rejection, bacterial infection, and poor osseointegration. Bioresorbable bone graft substitutes have emerged as attractive alternatives to conventional strategies because they can mimic the composition and mechanical properties of the native bone. Among these, bioactive glasses (BGs) hold great potential to be used as biomaterials for bone tissue engineering owing to their biomimetic composition and high biocompatibility and osteoinductivity. Here, we report the development of a novel composite biomaterial for bone tissue engineering based on the incorporation of a modified strontium‐ and lithium‐doped 58S BG (i.e., BG‐5/5) into gelatin methacryloyl (GelMA) hydrogels. We characterized the physicochemical properties of the BG formulation via different analytical techniques. Composite hydrogels were then prepared by directly adding BG‐5/5 to the GelMA hydrogel precursor, followed by photocrosslinking of the polymeric network via visible light. We characterized the physical, mechanical, and adhesive properties of GelMA/BG‐5/5 composites, as well as their in vitro cytocompatibility and osteoinductivity. In addition, we evaluated the antimicrobial properties of these composites in vitro, using a strain of methicillin‐resistant Staphylococcus Aureus. GelMA/BG‐5/5 composites combined the functional characteristics of the inorganic BG component, with the biocompatibility, biodegradability, and biomimetic composition of the hydrogel network. This novel biomaterial could be used for developing osteoinductive scaffolds or implant surface coatings with intrinsic antimicrobial properties and higher therapeutic efficacy.

  • Transcriptome dynamics of long noncoding RNAs and transcription factors demarcate human neonatal, adult, and human mesenchymal stem cell‐derived engineered cartilage
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2019-12-18
    Daniel J. Vail; Rodrigo A. Somoza; Arnold I. Caplan; Ahmad M. Khalil

    The engineering of a native‐like articular cartilage (AC) is a long‐standing objective that could serve the clinical needs of millions of patients suffering from osteoarthritis and cartilage injury. An incomplete understanding of the developmental stages of AC has contributed to limited success in this endeavor. Using next generation RNA sequencing, we have transcriptionally characterized two critical stages of AC development in humans—that is, immature neonatal and mature adult, as well as tissue‐engineered cartilage derived from culture expanded human mesenchymal stem cells. We identified key transcription factors (TFs) and long noncoding RNAs (lncRNAs) as candidate drivers of the distinct phenotypes of these tissues. AGTR2, SCGB3A1, TFCP2L1, RORC, and TBX4 stand out as key TFs, whose expression may be capable of reprogramming engineered cartilage into a more expandable and neonatal‐like cartilage primed for maturation into biomechanically competent cartilage. We also identified that the transcriptional profiles of many annotated but poorly studied lncRNAs were dramatically different between these cartilages, indicating that lncRNAs may also be playing significant roles in cartilage biology. Key neonatal‐specific lncRNAs identified include AC092818.1, AC099560.1, and KC877982. Collectively, our results suggest that tissue‐engineered cartilage can be optimized for future clinical applications by the specific expression of TFs and lncRNAs.

  • Tubular Scaffold with Microchannels and an H‐shaped Lumen Loaded with BMSCs Promotes Neuroregeneration and Inhibits Apoptosis after Spinal Cord Injury
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2019-12-10
    Xue Chen, Jian Wu, Rongcheng Sun, Yahong Zhao, Yi Li, Jingying Pan, Ying Chen, Xiaodong Wang

    As a result of its complex histological structure, regeneration patterns of gray and white matter are quite different in the spinal cord. Therefore, tissue engineering scaffolds for repairing spinal cord injury must be able to adapt to varying neural regeneration patterns. The aim of the present study was to improve a previously reported spinal cord‐mimicking partition‐type scaffold by adding microchannels on a single tubular wall along its longitudinal axis, thus integrating the two architectures of a single H‐shaped central tube and many microchannels. Next, the integrated scaffold was loaded with bone marrow stromal cells (BMSCs) and transplanted to bridge the 5‐mm defect of a complete transverse lesion in the thoracic spinal cord of rats. Subsequently, effects on nerve regeneration, locomotion function recovery, and early neuroprotection were observed. After 1 year of repair, the integrated scaffold could guide the regeneration of axons appearing in the debris of degraded microchannels, especially serotonin receptor 1A receptor‐positive axonal tracts, which were relatively orderly arranged. Moreover, a network of nerve fibers was present and a few BMSCs expressed neuronal markers in tubular lumens. Functionally, electrophysiological and locomotor functions of rats were partially recovered. In addition, we found that BMSCs could protect neurons and oligodendrocytes from apoptosis during the early stage of implantation. Taken together, our results demonstrate the potential of this novel integrated scaffold loaded with BMSCs to promote spinal cord regeneration through mechanical guidance and neuroprotective mechanisms.

  • Effect of mechanical loading and substrate elasticity on the osteogenic and adipogenic differentiation of mesenchymal stem cells
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2019-12-07
    Hatice Imran Gungordu, Min Bao, Sjoerd van Helvert, John A. Jansen, Sander C.G. Leeuwenburgh, X. Frank Walboomers

    Mesenchymal stem cells (MSCs) are highly sensitive to biomechanics of their extracellular environment. Generally, a higher elasticity of culture substrates can drive cells into the osteogenic lineage, whereas low substrate elasticity results in adipogenesis. Applied mechanical loading by cyclic strain is another major variable influencing cell fate. Yet, little is known about the simultaneous effect of both cues. Therefore, the present study investigated the relative importance of both cues on differentiation. MSCs were cultured in an osteogenic and also an adipogenic environment on soft polyacrylamide (PAAm; E = 23 ± 0.3 kPa), stiff PAAm (111 ± 2 kPa), and polydimethylsiloxane (PDMS; E = 1,5 ± 0.07 MPa) either unstrained or with 8% cyclic strain at 1 Hz. Without strain, the relative expression of the early osteogenic marker alkaline phosphatase (ALP) was significantly higher (78%) on PDMS than on both PAAm. With 8% cyclic strain, ALP expression increased for all groups in comparison with unstrained controls. The highest increase was observed for the soft PAAm by 36%. Moreover, relative oil red O (ORO) expression—indicating adipogenesis—was the highest for unstrained soft PAAm. On the other hand, the percentage of ORO positive cells significantly decreased by 57% and 69% for soft and stiff PAAm when strained. In conclusion, biomaterial elasticity and mechanical loading can act simultaneously on cell differentiation. Substrate elasticity is an important factor, regulating the differentiation, but cyclic strain can drive MSCs towards the osteogenesis even on the softest substrate. As such, the osteogenic effect of mechanical loading can overrule the adipogenic effect of soft substrates, thereby acting as an inhibitor.

  • The mechanobiology of tendon fibroblasts under static and uniaxial cyclic load in a 3D tissue engineered model mimicking native extracellular matrix
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2019-12-03
    Prasad Sawadkar, Darren Player, Laurent Bozec, Vivek Mudera

    Tendon mechanobiology plays a vital role in tendon repair and regeneration; however, this mechanism is currently poorly understood. We tested the role of different mechanical loads on extracellular matrix (ECM) remodelling gene expression and the morphology of tendon fibroblasts in collagen hydrogels, designed to mimic native tissue. Hydrogels were subjected to precise static or uniaxial loading patterns of known magnitudes and sampled to analyse gene expression of known mechano‐responsive ECM‐associated genes (Collagen I, Collagen III, Tenomodulin, and TGF‐β). Tendon fibroblast cytomechanics was studied under load by using a tension culture force monitor, with immunofluorescence and immunohistological staining used to examine cell morphology. Tendon fibroblasts subjected to cyclic load showed that endogenous matrix tension was maintained, with significant concomitant upregulation of ECM remodelling genes, Collagen I, Collagen III, Tenomodulin, and TGF‐β when compared with static load and control samples. These data indicate that tendon fibroblasts acutely adapt to the mechanical forces placed upon them, transmitting forces across the ECM without losing mechanical dynamism. This model demonstrates cell‐material (ECM) interaction and remodelling in preclinical a platform, which can be used as a screening tool to understand tendon regeneration.

  • Mesenchymal stem cells in chemotherapy‐induced peripheral neuropathy: A new challenging approach that requires further investigations
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2019-12-02
    Khaled F. Al‐Massri, Lamiaa A. Ahmed, Hanan S. El‐Abhar

    Chemotherapeutic drugs may disrupt the nervous system and cause chemotherapy‐induced peripheral neuropathy (CIPN) as side effects. There are no completely successful medications for the prevention or treatment of CIPN. Many drugs such as tricyclic antidepressants and anticonvulsants have been used for symptomatic treatment of CIPN. Unfortunately, these drugs often give only partial relief or have dose‐limiting side effects. Thus, the treatment of CIPN became a challenge because of failure to regenerate and repair the injured neurons. Mesenchymal stem cell (MSC) therapy is a new attractive approach for CIPN. Evidence has demonstrated that MSCs play important roles in reducing oxidative stress, neuroinflammation, and apoptosis, as well as mediating axon regeneration after nerve damage in several experimental studies and some clinical trials. We will briefly review the pathogenesis of CIPN, traditional therapies used and their drawbacks as well as therapeutic effects of MSCs, their related mechanisms, future challenges for their clinical application, and the additional benefit of their combination with pharmacological agents. MSCs‐based therapies may provide a new therapeutic strategy for patients suffering from CIPN where further investigations are required for studying their exact mechanisms. Combined therapy with pharmacological agents can provide another promising option for enhancing MSC therapy success while limiting its adverse effects.

  • Composite clinoptilolite/PCL‐PEG‐PCL scaffolds for bone regeneration: In vitro and in vivo evaluation
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2019-12-02
    Ahmet Engin Pazarçeviren, Tayfun Dikmen, Korhan Altunbaş, Volkan Yaprakçı, Özge Erdemli, Dilek Keskin, Ayşen Tezcaner

    In this study, clinoptilolite (CLN) was employed as a reinforcement in a polymer‐based composite scaffold in bone tissue engineering and evaluated in vivo for the first time. Highly porous, mechanically stable, and osteogenic CLN/PCL‐PEG‐PCL (CLN/PCEC) scaffolds were fabricated with modified particulate leaching/compression molding technique with varying CLN contents. We hypothesized that CLN reinforcement in a composite scaffold will improve bone regeneration and promote repair. Therefore, the scaffolds were analyzed for compressive strength, biodegradation, biocompatibility, and induction of osteogenic differentiation in vitro. CLN inclusion in PC‐10 (10% w/w) and PC‐20 (20% w/w) scaffolds revealed 54.7% and 53.4% porosity, higher dry (0.62 and 0.76 MPa), and wet (0.37 and 0.45 MPa) compressive strength, greater cellular adhesion, alkaline phosphatase activity (2.20 and 2.82 mg/gDNA/min), and intracellular calcium concentration (122.44 and 243.24 g Ca/mgDNA). The scaffolds were evaluated in a unicortical bone defect at anterior aspect of proximal tibia of adult rabbits 4 and 8 weeks postimplantation. Similar to in vitro results, CLN‐containing scaffolds led to efficient regeneration of bone in a dose‐dependent manner. PC‐20 demonstrated highest quality of bone union, cortex development, and bone‐scaffold interaction at the defect site. Therefore, higher CLN content in PC‐20 permitted robust remodeling whereas pure PCEC (PC‐0) scaffolds displayed fibrous tissue formation. Consequently, CLN was proven to be a potent reinforcement in terms of promoting mechanical, physical, and biological properties of polymer‐based scaffolds in a more economical, easy‐to‐handle, and reproducible approach.

  • A composite Gelatin/hyaluronic acid hydrogel as an ECM mimic for developing mesenchymal stem cell‐derived epithelial tissue patches
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2019-12-02
    Pramod Kumar, Sait Ciftci, Julien Barthes, Helena Knopf‐Marques, Céline Blandine Muller, Christian Debry, Nihal E. Vrana, Amir M. Ghaemmaghami

    Here we report fabrication of Gelatin‐based biocomposite films and their application in developing epithelial patches. The films were loaded with an epithelial cell growth factor cocktail and used as an extracellular matrix mimic for in vitro regeneration of organized respiratory epithelium using Calu‐3 cell line and mesenchymal stem cells (MSCs). Our data show differentiation of Calu‐3 cells on composite films as evidenced by tight junction protein expression and barrier formation. The films also supported attachment, migration, and proliferation of alveolar basal epithelial cell line A549. We also show the suitability of the composite films as a biomimetic scaffold and growth factor delivery platform for differentiation of human MSCs to epithelial cells. MSCs differentiation to the epithelial lineage was confirmed by staining for epithelial and stem cell specific markers. Our data show that the MSCs acquire the epithelial characteristics after 2 weeks with significant reduction in vimentin, increase in pan cytokeratin expression, and morphological changes. However, despite the expression of epithelial lineage markers, these cells did not form fully functional tight junctions as evidenced by low expression of junctional protein ZO1. Further optimisation of culture conditions and growth factor cocktail is required to enhance tight junction formation in MSCs‐derived epithelial cells on the composite hydrogels. Nevertheless, our data clearly highlight the possibility of using MSCs in epithelial tissue engineering and the applicability of the composite hydrogels as transferrable extracellular matrix mimics and delivery platforms with potential applications in regenerative medicine and in vitro modelling of barrier tissues.

  • Bioengineering and in utero transplantation of fetal skin in the sheep model: A crucial step towards clinical application in human fetal spina bifida repair
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2019-11-29
    Luca Mazzone, Ueli Moehrlen, Nicole Ochsenbein‐Kölble, Luca Pontiggia, Thomas Biedermann, Ernst Reichmann, Martin Meuli

    An intricate problem during open human fetal surgery for spina bifida regards back skin closure, particularly in those cases where the skin defect is much too large for primary closure. We hypothesize that tissue engineering of fetal skin might provide an adequate autologous skin substitute for in utero application in such situations. Eight sheep fetuses of four time‐mated ewes underwent fetoscopic skin biopsy at 65 days of gestation. Fibroblasts and keratinocytes isolated from the biopsy were used to create fetal dermo‐epidermal skin substitutes. These were transplanted on the fetuses by open fetal surgery at 90 days of gestation on skin defects (excisional wounds) created during the same procedure. Pregnancy was allowed to continue until euthanasia at 120 days of gestation. The graft area was analyzed macroscopically and microscopically. The transplanted fetal dermo‐epidermal skin substitutes was well discernable in situ in three of the four fetuses available for analysis. Histology confirmed healed grafts with a close to natural histological skin architecture four weeks after in utero transplantation. This experimental study generates evidence that laboratory grown autologous fetal skin analogues can successfully be transplanted in utero. These results have clinical implications as an analogous procedure might be applied in human fetuses undergoing prenatal repair to facilitate primary skin closure. Finally, this study may also fertilize the field of fetal tissue engineering in general, particularly when more interventional, minimally invasive, and open fetal surgical procedures become available.

  • Construction of a dermis–fat composite in vivo: Optimizing heterogeneous acellular dermal matrix with in vitro pretreatment
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2019-11-27
    Zhu Zhu, Zhao‐Qi Yuan, Cheng Huang, Rui Jin, Di Sun, Jun Yang, Xu‐Song Luo

    Dermis–fat composite tissues have been widely used in plastic and reconstructive surgery and were previously constructed using hydrogel‐type scaffolds. The constructs can be used for in vitro cosmetic and pharmaceutical testing but are not mechanically strong enough for in vivo applications. In this study, we used heterogeneous (porcine) acellular dermal matrix (PADM) as dermal layer scaffold. PADM was pretreated with the laser micropore technique and then precultured with rat adipose‐derived stem cells (rADSCs) in vitro. rADSCs proliferated well on pretreated/unpretreated PADM, showing increased expression of genes associated with inflammatory regulation, proangiogenesis, and stemness, indicating that pretreated/unpretreated PADM both provide a beneficial microenvironment for rADSCs to exert their paracrine function. After in vitro processing, the rADSCs–polyporous PADM and PADM without pretreatments were implanted into the back of rats respectively, followed by adipose tissue transplantation. After implantation, the inflammation induced by pretreated PADM was significantly attenuated and localized compared to the unpretreated group. Moreover, the vascularization was faster, and more adipose tissue was formed in the pretreated group. Sound dermis–fat composite tissue was constructed with sufficient strength, which can potentially be used for actual repair application.

  • Nerve lengthening and subsequent end‐to‐end repair yield more favourable outcomes compared with autograft repair of rat sciatic nerve defects
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2019-11-26
    Holly M. Howarth, Adarsh Kadoor, Rayeheh Salem, Brogan Nicolds, Stephanie Adachi, Achilles Kanaris, Richard M. Lovering, Justin M. Brown, Sameer B. Shah

    Outcomes of end‐to‐end nerve repairs are more successful compared with outcomes of repairs bridged by nerve grafts. However, end‐to‐end repairs are not always possible for large nerve gaps, as excessive tension may cause catastrophic failure. In this study, we built on previous nerve‐lengthening studies to test the hypotheses that gradual lengthening of the proximal stump across a large nerve gap enables an end‐to‐end repair and such a repair results in more favourable regenerative outcomes than autografts, which represent the gold standard in bridging nerve gaps. To test these, we compared structural and functional outcomes in Lewis rats after repair of sciatic nerve gaps using either autografts or a novel compact internal fixator device, which was used to lengthen proximal nerve stumps towards the distal stump over 2 weeks, prior to end‐to‐end repair. Twelve weeks after the initial injury, outcomes following nerve lengthening/end‐to‐end repair were either comparable or superior in every measure compared with repair by autografting. The sciatic functional index was not significantly different between groups at 12 weeks. However, we observed a reduced rate of contracture and corresponding significant increase in paw length in the lengthening group. This functional improvement was consistent with structural regeneration; axonal growth distal to the injury was denser and more evenly distributed compared with the autograft group, suggesting substantial regeneration into both tibial and peroneal branches of the sciatic nerve. Our findings show that end‐to‐end repairs following nerve lengthening are possible for large gaps and that this strategy may be superior to graft‐based repairs.

  • Comparison between calcium carbonate and β‐tricalcium phosphate as additives of 3D printed scaffolds with polylactic acid matrix
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2019-11-26
    Ricardo Donate, Mario Monzón, Zaida Ortega, Ling Wang, Viviana Ribeiro, David Pestana, Joaquim M. Oliveira, Rui L. Reis

    In this study, polylactic acid (PLA)‐based composite scaffolds with calcium carbonate (CaCO3) and beta‐tricalcium phosphate (β‐TCP) were obtained by 3D printing. These structures were evaluated as potential 3D structures for bone tissue regeneration. Morphological, mechanical, and biological tests were carried out in order to compare the effect of each additive (added in a concentration of 5% w/w) and the combination of both (2.5% w/w of each one), on the PLA matrix. The scaffolds manufactured had a mean pore size between 400–425 μm and a porosity value in the range of 50–60%. According to the results, both additives promoted an increase of the porosity, hydrophilicity, and surface roughness of the scaffolds, leading to a significant improvement of the metabolic activity of human osteoblastic osteosarcoma cells. The best results in terms of cell attachment after 7 days were obtained for the samples containing CaCO3 and β‐TCP particles due to the synergistic effect of both additives, which results in an increase in osteoconductivity and in a microporosity that favours cell adhesion. These scaffolds (PLA:CaCO3:β‐TCP 95:2.5:2.5) have suitable properties to be further evaluated for bone tissue engineering applications.

  • Growth factor‐free, angiogenic hydrogel based on hydrophobically modified Alaska pollock gelatin
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2019-11-19
    Yosuke Mizuno, Tetsushi Taguchi

    Angiogenesis is important for supplying oxygen and nutrients to implanted cells and organs and thereby promoting their survival. However, exogenously administered growth factors such as vascular endothelial growth factor (VEGF) have a short half‐life and are unstable under physiological conditions. In the present study, we developed an angiogenesis—inducing hydrogel by modifying Alaska pollock‐derived gelatin with a dodecyl group (C12‐ApGltn), and demonstrated that it is biodegradable and highly fluid at room temperature (25°C). C12‐ApGltn dissolved in phosphate‐buffered saline at 20 w/v% formed a self‐assembling hydrogel with thixotropic properties that stimulated VEGF secretion by macrophage‐like RAW264 cells. Moreover, C12‐ApGltn stimulated nuclear factor‐κB and VEGF expression when subcutaneously injected into mice and increased the cluster of differentiation 31‐positive area compared with injection of unmodified ApGltn and phosphate‐buffered saline control in the absence of any growth factors. Hematoxylin and eosin staining confirmed vascular capillaries around the C12‐ApGltn injection site. These results demonstrate that C12‐ApGltn hydrogel is a promising angiogenic material for clinical applications that can stimulate endogenous VEGF expression without requiring additional growth factors.

  • Understanding the multifaceted mechanisms of diabetic wound healing and therapeutic application of stem cells conditioned medium in the healing process
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2019-11-19
    Lai Woon Fui, Michael Phang Weng Lok, Vijayendran Govindasamy, Then Kong Yong, Then Khong Lek, Anjan Kumar Das

    Mesenchymal stem cells (MSCs) transplantation seems to be a promising new therapy for diabetic wound healing (DWH), and currently, arrays of MSCs from various sources ranging from umbilical, adipose to dental sources are available as a treatment modality for this disease. However, it now appears that only a fraction of transplanted cells actually assimilate and survive in host tissues suggesting that the major mechanism by which stem cells participate in tissue repair are most likely related to their secretome level. These include a wide range of growth factors, cytokines, and chemokines, which can be found from the conditioned medium (CM) used to culture the cells. Basic studies and preclinical work confirm that the therapeutic effect of CMs are comparable with the application of stem cells. This review describes in detail the wound healing process in diabetes and the cellular and biological factors that influence the process. Subsequently, through a comprehensive literature search of studies related to wound healing in diabetics, we aim to provide an overview of scientific merits of using MSCs‐CM in the treatment of diabetic wound as well as the significant caveats, which restricts its potential use in clinical set‐ups. To our best knowledge, this is one of the first review papers that collect the importance of stem cells as an alternative treatment to the DWH. We anticipate that the success of this treatment will have a significant clinical impact on diabetic wounds.

  • Impact of ultraviolet radiation on dermal and epidermal DNA damage in a human pigmented bilayered skin substitute
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2019-11-17
    Benjamin Goyer, Ulysse Pereira, Brice Magne, Danielle Larouche, Sélia Kearns‐Turcotte, Patrick J. Rochette, Ludovic Martin, Lucie Germain

    Our laboratory has developed a scaffold‐free cell‐based method of tissue engineering to produce bilayered tissue‐engineered skin substitutes (TESs) from epidermal and dermal cells. However, TES pigmentation is absent or heterogeneous after grafting, due to a suboptimal number of melanocytes in culture. Our objectives were to produce TESs with a sufficient quantity of melanocytes from different pigmentation phototypes (light and dark) to achieve a homogeneous color and to evaluate whether the resulting pigmentation was photoprotective against ultraviolet radiation (UVR)‐induced DNA damage in the dermis and the epidermis. TESs were cultured using different concentrations of melanocytes (100, 200, and 1,500 melanocytes/mm2), and pigmentation was evaluated in vitro and after grafting onto an athymic mouse excisional model. Dermal and epidermal DNA damage was next studied, exposing pigmented TESs to 13 and 32.5 J/cm2 UVR in vitro. We observed that melanocyte cell density increased with culture time until reaching a plateau corresponding to the cell distribution of native skin. Pigmentation of melanocyte‐containing TESs was similar to donor skin, with visible melanin transfer from melanocytes to keratinocytes. The amount of melanin in TESs was inversely correlated to the UVR‐induced formation of cyclobutane pyrimidine dimer in dermal fibroblasts and keratinocytes. Our results indicate that the pigmentation conferred by the addition of melanocytes in TESs protects against UVR‐induced DNA damage. Therefore, autologous pigmented TESs could ensure photoprotection after grafting.

  • In vivo implantation of a tissue engineered stem cell seeded hemi‐laryngeal replacement maintains airway, phonation, and swallowing in pigs
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2017-12-17
    P. Herrmann, T. Ansari, A. Southgate, A. Varanou Jenkins, L. Partington, C. Carvalho, S. Janes, M. Lowdell, P.D. Sibbons, M.A. Birchall

    Laryngeal functional impairment relating to swallowing, vocalisation, and respiration can be life changing and devastating for patients. A tissue engineering approach to regenerating vocal folds would represent a significant advantage over current clinical practice.

  • Evaluating the cytocompatibility and differentiation of bone progenitors on electrospun zein scaffolds
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2019-11-15
    Jessica Cardenas Turner, George Collins, Elizabeth A. Blaber, Eduardo A.C. Almeida, Treena L. Arinzeh

    Bone fractures often result in complications that require surgical intervention to promote fracture healing. Tissue engineering seeks to alleviate the need for autologous bone grafting by utilizing scaffolds that can promote bone fracture healing. Plant‐derived materials are desirable biomaterials because of their biodegradability, availability, and low immunogenicity. Among various plant‐derived proteins, zein, which is a corn protein, has shown promise for bone repair. However, when processed, zein is often blended with synthetic materials to improve mechanical properties and overall hydrolytic stability. In this study, pure zein was electrospun to create fibrous scaffolds and cross‐linked with trimethylolpropane triglycidyl ether to improve hydrolytic stability. Scaffolds were characterized and evaluated in vitro for promoting the osteogenic differentiation of MC3T3‐E1 cells, which are bone progenitor cells. Cross‐linked zein scaffolds retained their uniform fiber morphologies after hydration. MC3T3‐E1 cells grew and differentiated on the zein scaffolds even in the absence of induction factors, as demonstrated by increased alkaline phosphatase activity, mineralization, and early upregulation of Runx2 gene expression, a transcription factor associated with osteoblast differentiation. These studies demonstrate that stable, zein fibrous scaffolds could have potential for use in bone repair applications.

  • Fabrication of tissue‐engineered cell sheets by automated cell culture equipment
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2019-11-14
    Ayako Nishimura, Ryota Nakajima, Ryo Takagi, Guangbin Zhou, Daisuke Suzuki, Masaharu Kiyama, Takayuki Nozaki, Toshiyuki Owaki, Tomomi Takahara, Shigeru Nagai, Taku Nakamura, Masakazu Sugaya, Koichi Terada, Yumiko Igarashi, Hiroko Hanzawa, Teruo Okano, Tatsuya Shimizu, Masayuki Yamato, Shizu Takeda

    Most cells for regenerative medicine are currently cultured manually. In order to promote the widespread use of regenerative medicine, it will be necessary to develop automated culture techniques so that cells can be produced in greater quantities at lower cost and with more stable quality. In the field of regenerative medicine technology, cell sheet therapy is an effective tissue engineering technique whereby cells can be grafted by attaching them to a target site. We have developed automated cell culture equipment to promote the use of this cell sheet regenerative treatment. This equipment features a fully closed culture vessel and circuit system that avoids contamination with bacteria and the like from the external environment, and it was designed to allow 10 cell sheets to be simultaneously cultured in parallel. We used this equipment to fabricate 50 sheets of human oral mucosal epithelial cells in five automated culture tests in this trial. By analyzing these sheets, we confirmed that 49 of the 50 sheets satisfied the quality standards of clinical research. To compare the characteristics of automatically fabricated cell sheets with those of manually fabricated cell sheets, we performed histological analyses using immunostaining and transmission electron microscopy. The results confirmed that cell sheets fabricated with the automated cell culture are differentiated in the same way as cultures fabricated manually.

  • Extracellular matrix cues modulate Schwann cell morphology, proliferation, and protein expression
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2019-11-14
    Zhenyuan Xu, Jacob A. Orkwis, Braden M. DeVine, Greg M. Harris

    Peripheral nerve injuries require a complex set of signals from cells, macrophages, and the extracellular matrix (ECM) to induce regeneration across injury sites and achieve functional recovery. Schwann cells (SCs), the major glial cell in the peripheral nervous system (PNS), are critical to nerve regeneration due to their inherent capacity for altering phenotype postinjury to facilitate wound healing. The ECM plays a vital role in wound healing as well as regulating cell phenotype during tissue repair. To examine the underlying mechanisms between the ECM and SCs, this work sought to determine how specific ECM cues regulate the phenotype of SCs. To address this, SCs were cultured on polydimethylsiloxane substrates of a variable Young's modulus coated with ECM proteins. Cells were analyzed for spreading area, proliferation, cell and nuclear shape, and c‐Jun expression. It was found that substrates with a stiffness of 8.67 kPa coated with laminin promoted the highest expression of c‐Jun, a marker signifying a “regenerative” SC. Microcontact printed, cell adhesive areas were then utilized to precisely control the geometry and spreading of SCs and by controlling spreading area and cellular elongation; expression of c‐Jun was either promoted or downregulated. These results begin to address the significant interplay between ECM cues and phenotype of SCs, while offering a potential means to enhance PNS regeneration through cellular therapies.

  • Chorionic and amniotic placental membrane‐derived stem cells, from gestational diabetic women, have distinct insulin secreting cell differentiation capacities
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2019-11-14
    Liyun Chen, Nicholas R. Forsyth, Pensee Wu

    Women with gestational diabetes mellitus (GDM), and their offspring, are at high risk of developing type 2 diabetes. Chorionic (CMSCs) and amniotic mesenchymal stem cells (AMSCs) derived from placental membranes provide a source of autologous stem cells for potential diabetes therapy. We established an approach for the CMSC/AMSC‐based generation of functional insulin‐producing cells (IPCs). CMSCs/AMSCs displayed significantly elevated levels of NANOG and OCT4 versus bone marrow‐derived MSCs, indicating a potentially broad differentiation capacity. Exposure of Healthy‐ and GDM‐CMSCs/AMSCs to long‐term high‐glucose culture resulted in significant declines in viability accompanied by elevation, markedly so in GDM‐CMSCs/AMSCs, of senescence/stress markers. Short‐term high‐glucose culture promoted pancreatic transcription factor expression when coupled to a 16‐day step‐wise differentiation protocol; activin A, retinoic acid, epidermal growth factor, glucagon‐like peptide‐1 and other chemical components, generated functional IPCs from both Healthy‐ and GDM‐CMSCs. Healthy‐/GDM‐AMSCs displayed betacellulin‐sensitive insulin expression, which was not secreted upon glucose challenge. The pathophysiological state accompanying GDM may cause irreversible impairment to endogenous AMSCs; however, GDM‐CMSCs possess comparable therapeutic potential with Healthy‐CMSCs and can be effectively reprogrammed into insulin‐secreting cells.

  • Octacalcium phosphate collagen composite stimulates the expression and activity of osteogenic factors to promote bone regeneration
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2019-11-12
    Atsumu Kouketsu, Keiko Matsui, Tadashi Kawai, Yushi Ezoe, Toshiki Yanagisawa, Ayato Yasuda, Tetsu Takahashi, Shinji Kamakura

    This study investigated the bone regenerative properties of an octacalcium phosphate collagen composite (OCP/Col) in a rat calvarial bone defect model.

  • Optimising platelet secretomes to deliver robust tissue‐specific regeneration
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2019-11-11
    David Scully, Peggy Sfyri, Holly N. Wilkinson, Andrea Acebes‐Huerta, Sandrine Verpoorten, María Carmen Muñoz‐Turrillas, Andrew Parnell, Ketan Patel, Matthew J. Hardman, Laura Gutiérrez, Antonios Matsakas

    Promoting cell proliferation is the cornerstone of most tissue regeneration therapies. As platelet‐based applications promote cell division and can be customised for tissue‐specific efficacy, this makes them strong candidates for developing novel regenerative therapies. Therefore, the aim of this study was to determine if platelet releasate could be optimised to promote cellular proliferation and differentiation of specific tissues. Growth factors in platelet releasate were profiled for physiological and supraphysiological platelet concentrations. We analysed the effect of physiological and supraphysiological releasate on C2C12 skeletal myoblasts, H9C2 rat cardiomyocytes, human dermal fibroblasts (HDF), HaCaT keratinocytes, and chondrocytes. Cellular proliferation and differentiation were assessed through proliferation assays, mRNA, and protein expression. We show that supraphysiological releasate is not simply a concentrated version of physiological releasate. Physiological releasate promoted C2C12, HDF, and chondrocyte proliferation with no effect on H9C2 or HaCaT cells. Supraphysiological releasate induced stronger proliferation in C2C12 and HDF cells compared with physiological releasate. Importantly, supraphysiological releasate induced proliferation of H9C2 cells. The proliferative effects of skeletal and cardiac muscle cells were in part driven by vascular endothelial growth factor alpha. Furthermore, supraphysiological releasate induced differentiation of H9C2 and C2C12, HDF, and keratinocytes. This study provides insights into the ability of releasate to promote muscle, heart, skin, and cartilage cell proliferation and differentiation and highlights the importance of optimising releasate composition for tissue‐specific regeneration.

  • Immunomodulatory effect of human bone marrow‐derived mesenchymal stromal/stem cells on peripheral blood T cells from rheumatoid arthritis patients
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2019-11-11
    Mónia Pedrosa, Joana Gomes, Paula Laranjeira, Cátia Duarte, Susana Pedreiro, Brígida Antunes, Tânia Ribeiro, Francisco Santos, António Martinho, Margarida Fardilha, M. Rosário Domingues, Manuel Abecasis, José António P. da Silva, Artur Paiva

    Rheumatoid arthritis (RA) is a Th1/Th17‐mediated autoimmune disease whose current treatment, consisting in the blockage of inflammatory cytokines by disease‐modifying antirheumatic drugs, is not effective for all patients. The therapeutic potential of mesenchymal stromal/stem cells' (MSCs) immunomodulatory properties is being explored in RA. Here, we investigate the effect of human bone marrow (BM)‐MSCs on the expression of cytokines involved in RA physiopathology by the distinct functional compartments of CD4+ and CD8+ T cells from RA patients. Peripheral blood mononuclear cells from healthy individuals (n = 6) and RA patients (n = 12) were stimulated with phorbol myristate acetate plus ionomycin and cultured in the presence/absence of BM‐MSCs. The expression of (interleukin) IL‐2, tumor necrosis factor alpha (TNF‐α), and interferon‐gamma (IFN‐γ) was evaluated in naive, central memory, effector memory, and effector CD4+ and CD8+ T cells, whereas IL‐6, IL‐9, and IL‐17 expression was measured in total CD4+ and CD8+ T cells. mRNA expression of IL‐4, IL‐10, transforming growth factor beta (TGF‐β), cytotoxic T‐lymphocyte‐associated antigen 4, and/or forkhead box P3 was quantified in fluorescence‐activated cell sorting‐purified CD4+ T cells, CD8+ T cells, and CD4+ Treg. BM‐MSCs inhibited the production of TNF‐α, IL‐17, IL‐6, IL‐2, IFN‐γ, and IL‐9 by T cells from RA patients, mainly by reducing the percentage of cells producing cytokines. This inhibitory effect was transversal to all T cell subsets analyzed. At mRNA level, BM‐MSCs increased expression of IL‐10 and TGF‐β by CD4+ and CD8+ T cells. BM‐MSCs displayed a striking inhibitory action over T cells from RA patients, reducing the expression of cytokines involved in RA physiopathology. Remarkably, BM‐MSC‐derived immunomodulation affected either naive, effector, and memory T cells.

  • Effects of calcium concentration on nonviral gene delivery to bone marrow‐derived stem cells
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2019-11-11
    Timothy M. Acri, Noah Z. Laird, Sean M. Geary, Aliasger K. Salem, Kyungsup Shin

    Calcium ions (Ca2+) influence natural bone healing, and calcium is frequently used in bone tissue engineering scaffolds and cements. Scaffolds can also incorporate gene delivery systems to further promote osteoblast differentiation. Thus, our goal was to identify if Ca2+ concentration affects the transfection of bone marrow stromal cells because these cells play a major role in bone healing and can infiltrate gene‐activated scaffolds designed to promote bone growth.

  • Directional topography gradients drive optimum alignment and differentiation of human myoblasts
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2019-11-10
    Ana Maria Almonacid Suarez, Qihui Zhou, Patrick van Rijn, Martin C. Harmsen

    Tissue engineering of skeletal muscle aims to replicate the parallel alignment of myotubes on the native tissue. Directional topography gradients allow the study of the influence of topography on cellular orientation, proliferation, and differentiation, resulting in yield cues and clues to develop a proper in vitro environment for muscle tissue engineering. In this study, we used a polydimethylsiloxane‐based substrate containing an aligned topography gradient with sinusoidal features ranging from wavelength (λ) = 1,520 nm and amplitude (A) =176 nm to λ = 9,934 nm and A = 2,168 nm. With this topography gradient, we evaluated the effect of topography on human myoblasts distribution, dominant orientation, cell area, nuclei coverage, cell area per number of nuclei, and nuclei area of myotubes. We showed that human myoblasts aligned and differentiated irrespective of the topography section. In addition, aligned human myotubes showed functionality and maturity by contracting spontaneously and nuclei peripheral organization resembling natural myotubes.

  • Autologous blood coagulum is a physiological carrier for BMP6 to induce new bone formation and promote posterolateral lumbar spine fusion in rabbits
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2019-11-10
    Slobodan Vukicevic, Lovorka Grgurevic, Igor Erjavec, Marko Pecin, Tatjana Bordukalo‐Niksic, Nikola Stokovic, Marija Lipar, Hrvoje Capak, Drazen Maticic, Reinhard Windhager, T. Kuber Sampath, Munish Gupta

    In the present study, we describe autologous blood coagulum (ABC) as a physiological carrier for BMP6 to induce new bone formation. Recombinant human BMP6 (rhBMP6), dispersed within ABC and formed as an autologous bone graft substitute (ABGS), was evaluated either with or without allograft bone particles (ALLO) in rat subcutaneous implants and in a posterolateral lumbar fusion (PLF) model in rabbits. ABGS induced endochondral bone differentiation in rat subcutaneous implants. Coating ALLO by ABC significantly decreased the formation of multinucleated foreign body giant cells (FBGCs) in implants, as compared with ALLO alone. However, addition of rhBMP6 to ABC/ALLO induced a robust endochondral bone formation with little or no FBGCs in the implant. In rabbit PLF model, ABGS induced new bone formation uniformly within the implant resulting in a complete fusion when placed between two lumbar transverse processes in the posterolateral gutter with an optimum dose of 100‐μg rhBMP6 per ml of ABC. ABGS containing ALLO also resulted in a fusion where the ALLO was replaced by the newly formed bone via creeping substitution. Our findings demonstrate for the first time that rhBMP6, with ABC as a carrier, induced a robust bone formation with a complete spinal fusion in a rabbit PLF model. RhBMP6 was effective at low doses with ABC serving as a physiological substratum providing a permissive environment by protecting against foreign body reaction elicited by ALLO.

  • Phospholipid‐induced silk fibroin hydrogels and their potential as cell carriers for tissue regeneration
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2019-11-10
    Chavee Laomeephol, Marta Guedes, Helena Ferreira, Rui L. Reis, Sorada Kanokpanont, Siriporn Damrongsakkul, Nuno M. Neves

    Silk fibroin (SF) hydrogels can be obtained via self‐assembly, but this process takes several days or weeks, being unfeasible to produce cell carrier hydrogels. In this work, a phospholipid, namely, 1,2‐dimyristoyl‐sn‐glycero‐3‐phospho‐(1′‐rac‐glycerol) sodium salt (DMPG), was used to induce and accelerate the gelation process of SF solutions. Due to the amphipathic nature and negative charge of DMPG, electrostatic and hydrophobic interactions between the phospholipids and SF chains will occur, inducing the structural transition of SF chains to the beta sheet and consequently a rapid gel formation is observed (less than 50 min). Moreover, the gelation time can be controlled by varying the lipid concentration. To assess the potential of the hydrogels as cell carriers, several mammalian cell lines, including L929, NIH/3T3, SaOS‐2, and CaSki, were encapsulated into the hydrogel. The silk‐based hydrogels supported the normal growth of fibroblasts, corroborating their cytocompatibility. Interestingly, an inhibition in the growth of cancer‐derived cell lines was observed. Therefore, DMPG‐induced SF hydrogels can be successfully used as a 3D platform for in situ cell encapsulation, opening promising opportunities in biomedical applications, such as in cell therapies and tissue regeneration.

  • Possibilities and limitations of electrospun chitosan‐coated polycaprolactone grafts for rotator cuff tear repair
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2019-11-10
    Elmar Willbold, Mathias Wellmann, Bastian Welke, Nina Angrisani, Sarah Gniesmer, Andreas Kampmann, Andrea Hoffmann, Dominik de Cassan, Henning Menzel, Anna Lena Hoheisel, Birgit Glasmacher, Janin Reifenrath

    Acute and chronic rotator cuff tears remain challenging for therapy. A wide range of therapeutic approaches were developed but re‐tears and postoperative complications occur regularly. Especially in elderly people, the natural regeneration processes are decelerated, and graft materials are often necessary to stabilize the tendon‐to‐bone attachment and to improve the healing process. We here investigated in a small animal model a newly developed electrospun polycaprolactone fiber implant coated with a chitosan‐polycaprolactone graft copolymer and compared these implants biomechanically and histologically with either a commercially available porous polyurethane implant (Biomerix 3D Scaffold) or suture‐fixed tendons. Fifty‐one rats were divided into three groups of 17 animals each. In the first surgery, the left infraspinatus tendons of all rats were detached, and the animals recovered for 4 weeks. In the second surgery, the tendons were fixed with suture material only (suture‐fixed group; n = 17), whereas in the two experimental groups, the tendons were fixed with suture material and the polyurethane implant (Biomerix scaffold group; n = 17) or the modified electrospun polycaprolactone fiber implant (CS‐g‐PCL scaffold group; n=17), respectively. The unaffected right infraspinatus tendons were used as native controls. After a recovery of 8 weeks, all animals were clinically inconspicuous. In 12 animals of each group, repaired entheses were biomechanically tested for force at failure, stiffness, and modulus of elasticity, and in five animals, repaired entheses were analyzed histologically. Biomechanically, all parameters did not differ statistically significant between both implant groups, and the entheses failed typically at the surgical site. However, with respect to the force at failure, the median values of the two implant groups were smaller than the median value of the suture‐fixed group. Histologically, the modified polycaprolactone fiber implant showed no acute inflammation processes, a good infiltration with cells, ingrowth of blood vessels and tendinous tissue, and a normal fibrous ensheathment. Further improvement of the implant material could be achieved by additional implementation of drug delivery systems. Therewith, the used CS‐g‐PCL fiber mat is a promising basic material to reach the goal of a clinically usable graft for rotator cuff tear repair.

  • A clinical‐grade acellular matrix for esophageal replacement
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2019-11-07
    Lousineh Arakelian, Clémentine Caille, Lionel Faivre, Laurent Corté, Patrick Bruneval, Sara Shamdani, Camille Flageollet, Patricia Albanese, Thomas Domet, Mohamed Jarraya, Niclas Setterblad, Sabrina Kellouche, Jérôme Larghero, Pierre Cattan, Valérie Vanneaux

    In pathologies of the esophagus such as esophageal atresia, cancers, and caustic injuries, methods for full thickness esophageal replacement require the sacrifice of healthy intra‐abdominal organs such as the stomach and the colon and are associated with high morbidity, mortality, and poor functional results. To overcome these problems, tissue engineering methods are developed to create a substitute with scaffolds and cells. The aim of this study was to develop a simple and safe decellularization process in order to obtain a clinical grade esophageal extracellular matrix. Following the decontamination step, porcine esophagi were decellularized in a bioreactor with sodium dodecyl sulfate and ethylenediaminetetraacetic acid for 3 days and were rinsed with deionized water. DNA was eliminated by a 3‐hr DNase treatment. To remove any residual detergent, the matrix was then incubated with an absorbing resin. The resulting porcine esophageal matrix was characterized by the assessment of the efficiency of the decellularization process (DNA quantification), evaluation of sterility and absence of cytotoxicity, and its composition and biomechanical properties, as well as the possibility to be reseeded with mesenchymal stem cells. Complete decellularization with the preservation of the general structure, composition, and biomechanical properties of the native esophageal matrix was obtained. Sterility was maintained throughout the process, and the matrix showed no cytotoxicity. The resulting matrix met clinical grade criteria and was successfully reseeded with mesenchymal stem cells..

  • 16 T high static magnetic field inhibits receptor activator of nuclear factor kappa‐Β ligand‐induced osteoclast differentiation by regulating iron metabolism in Raw264.7 cells
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2019-11-06
    Dandan Dong, Jiancheng Yang, Gejing Zhang, Ting Huyan, Peng Shang

    High static magnetic fields (HiSMFs) are usually defined as those SMFs with intensities ≥1 T. Although many studies have indicated that SMFs have positive effects on bone tissue, there were limited studies that investigate the effects of cells, including osteoclasts, to illustrate the effect of HiSMF on osteoclast differentiation, and whether iron involve in the altered osteoclast formation and resorption ability under HiSMF. 16 T HiSMF generated from a superconducting magnet was used. Osteoclastogenesis, bone resorption, acting ring formation, messenger ribonucleic acid expression, and protein expression were determined by tartrate‐resistant acid phosphatase staining, pits formation assay, rhodamine‐conjugated phalloidine staining, quantitative real‐time polymerase chain reaction, and western blot, respectively. The changes induced by HiSMF in the level of iron and the concentration of mitochondrial protein, adenosine triphosphate, reactive oxygen species, malonaldehyde, and glutathione were examined by atomic absorption spectrometry and corresponding commercial kits, respectively. The results showed that HiSMF significantly inhibited osteoclastic formation and resorption ability and reduced cellular iron content during osteoclast differentiation. Mitochondrial concentration and oxidative stress levels in osteoclasts were decreased under HiSMF. Mechanistically, HiSMF markedly blocked the expression of osteoclast‐associated transcription factors and osteoclast marker genes and inhibited iron absorption and iron storage‐related protein expression. These findings demonstrated that the effect of HiSMF on iron metabolism of osteoclasts was involved in the inhibition of HiSMF on osteoclast differentiation.

  • Evaluation of tenogenic differentiation potential of selected subpopulations of human adipose‐derived stem cells
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2019-11-06
    Ana I. Gonçalves, Dominika Berdecka, Márcia T. Rodrigues, Aysegul Dede Eren, Jan de Boer, Rui L. Reis, Manuela E. Gomes

    Identification of a suitable cell source and bioactive agents guiding cell differentiation towards tenogenic phenotype represents a prerequisite for advancement of cell‐based therapies for tendon repair. Human adipose‐derived stem cells (hASCs) are a promising, yet intrinsically heterogenous population with diversified differentiation capacities. In this work, we investigated antigenically‐defined subsets of hASCs expressing markers related to tendon phenotype or associated with pluripotency that might be more prone to tenogenic differentiation, when compared to unsorted hASCs. Subpopulations positive for tenomodulin (TNMD+ hASCs) and stage specific early antigen 4 (SSEA‐4+ hASCs), as well as unsorted ASCs were cultured up to 21 days in basic medium or media supplemented with TGF‐β3 (10 ng/ml), or GDF‐5 (50 ng/ml). Cell response was evaluated by analysis of expression of tendon‐related markers at gene level and protein level by real time RT‐PCR, western blot, and immunocytochemistry. A significant upregulation of scleraxis was observed for both subpopulations and unsorted hASCs in the presence of TGF‐β3. More prominent alterations in gene expression profile in response to TGF‐β3 were observed for TNMD+ hASCs. Subpopulations evidenced an increased collagen III and TNC deposition in basal medium conditions in comparison with unsorted hASCs. In the particular case of TNMD+ hASCs, GDF‐5 seems to influence more the deposition of TNC. Within hASCs populations, discrete subsets could be distinguished offering varied sensitivity to specific biochemical stimulation leading to differential expression of tenogenic components suggesting that cell subsets may have distinctive roles in the complex biological responses leading to tenogenic commitment to be further explored in cell based strategies for tendon tissues.

  • Improving intraoperative storage conditions for autologous bone grafts: An experimental investigation in mice
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2019-11-06
    Qiang Sun, Zhijun Li, Bo Liu, Xue Yuan, Shu Guo, Jill A. Helms

    Autologous bone grafts constitute the second most transplanted tissue in medicine today. The viability, and consequently the osteogenic capacity, of an autograft is directly impacted by the interval between harvest and transplantation, but how the temperature and the solution in which the graft is held intraoperatively affect viability is not clear. Using a syngeneic mouse model and in vivo bone‐forming assays, these variables were tested for their effects on programmed cell death, osteoprogenitor cell proliferation, and the ability of the autograft to ultimately produce new bone in an ectopic site. Based on these results, the intraoperative treatment with a WNT protein therapeutic was tested for its effects on the viability and osteogenic capacity of an autograft. Viability, programmed cell death, mitotic activity, osteogenic protein expression, and bone‐forming capacity were assessed. Experimental results demonstrated that the osteogenic capacity of an autograft is significantly improved by intraoperative storage in L‐WNT3A at physiological temperature.

  • Effect of adiponectin secreted from adipose‐derived stem cells on bone‐fat balance and bone defect healing
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2019-10-31
    Shimao Yang, Hanghang Liu, Yao Liu, Li Liu, Wenmei Zhang, En Luo

    The efficacy of adiponectin (APN) in regulating bone metabolism remains controversial. This study aimed to investigate the role of APN secreted from adipose‐derived stem cells on adipogenesis and osteogenesis. Human APN gene was transfected via recombinant adenovirus into adipose derived stem cells (ASCs) in vitro and were cocultured with bone marrow mesenchymal stem cells (BMSCs) in using a transwell chamber. Adipogenesis was inhibited in APN‐transfected ASCs; in BMSCs, adipogenesis was inhibited, but osteogenesis was promoted in coculture with APN‐transfected ASCs. Next, the same adenovirus construct was transfected into the abdominal adipose tissue of a Sprague Dawley rat in vivo, and then a tibia defect was established in the same rat. We confirmed there was higher gene and protein expression of APN in ASCs and the abdominal adipose tissue of these rat models. Development of adipocytes in abdominal adipose tissue was suppressed, and less new bone was formed in the bone defect area. In conclusion, APN secreted from ASCs could directly inhibit adipogenesis in ASCs and BMSCs and promote osteogenesis in the latter. However, APN overexpression in adipose tissue was inversely associated with bone formation in tibia defects potentially due to decreased levels of circulating bone‐activating hormones.

  • Corrigendum.
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2019-11-22

  • Trachea transplantation: from laboratory to patient.
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2015-06-09
    Claire Crowley,Martin Birchall,Alexander M Seifalian

    Today, tracheal lesions occupying<30%of the trachea in children and<50%in adults can be treated with primary resection, followed by end-to-end anastomosis. However, lesions larger than this require a tracheal replacement, of which there are currently few options available. The recent advancement of tissue-engineering principles in tracheal research is quickly opening up new vistas for airway reconstruction and creating a very promising future for medical science. This review discusses the main criteria required for the development of a tissue-engineered tracheal replacement. The criteria include: (a) appropriate cell types and sources; (b) biomolecules to direct the differentiation of the cells to the desired lineage; (c) a suitable scaffold for a cellular matrix; and (d) a bioreactor to facilitate cell attachment and proliferation and construct transport to theatre. Our group has designed and developed the world’s first synthetic tracheal replacement, using a novel nanocomposite material, also developed in our laboratory. It was implanted clinically in June 2011 with a successful outcome. The application of tissue-engineering approaches to tracheal replacement development is the first step towards the much-anticipated ‘off-the-shelf’ tissue-engineered technology, contributing extensively to the advancement in treatment and rehabilitation of patients afflicted with tracheal pathology.

  • Strategies for neurotrophin-3 and chondroitinase ABC release from freeze-cast chitosan-alginate nerve-guidance scaffolds.
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2014-06-04
    Nicola L Francis,Philipp M Hunger,Amalie E Donius,Ulrike G K Wegst,Margaret A Wheatley

    Freeze casting, or controlled unidirectional solidification, can be used to fabricate chitosan-alginate (C-A) scaffolds with highly aligned porosity that are suitable for use as nerve-guidance channels. To augment the guidance of growth across a spinal cord injury lesion, these scaffolds are now evaluated in vitro to assess their ability to release neurotrophin-3 (NT-3) and chondroitinase ABC (chABC) in a controlled manner. Protein-loaded microcapsules were incorporated into C-A scaffolds prior to freeze casting without affecting the original scaffold architecture. In vitro protein release was not significantly different when comparing protein loaded directly into the scaffolds with release from scaffolds containing incorporated microcapsules. NT-3 was released from the C-A scaffolds for 8 weeks in vitro, while chABC was released for up to 7 weeks. Low total percentages of protein released from the scaffolds over this time period were attributed to limitation of diffusion by the interpenetrating polymer network matrix of the scaffold walls. NT-3 and chABC released from the scaffolds retained bioactivity, as determined by a neurite outgrowth assay, and the promotion of neurite growth across an inhibitory barrier of chondroitin sulphate proteoglycans. This demonstrates the potential of these multifunctional scaffolds for enhancing axonal regeneration through growth-inhibiting glial scars via the sustained release of chABC and NT-3. Copyright © 2014 John Wiley & Sons, Ltd.

  • Large animal in vivo evaluation of a binary blend polymer scaffold for skeletal tissue-engineering strategies; translational issues.
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2015-02-19
    James O Smith,Edward R Tayton,Ferdous Khan,Alexander Aarvold,Richard B Cook,Allen Goodship,Mark Bradley,Richard O C Oreffo

    Binary blend polymers offer the opportunity to combine different desirable properties into a single scaffold, to enhance function within the field of tissue engineering. Previous in vitro and murine in vivo analysis identified a polymer blend of poly(l-lactic acid)-poly(ε-caprolactone) (PLLA:PCL 20:80) to have characteristics desirable for bone regeneration. Polymer scaffolds in combination with marrow-derived skeletal stem cells (SSCs) were implanted into mid-shaft ovine 3.5 cm tibial defects, and indices of bone regeneration were compared to groups implanted with scaffolds alone and with empty defects after 12 weeks, including micro-CT, mechanical testing and histological analysis. The critical nature of the defect was confirmed via all modalities. Both the scaffold and scaffold/SSC groups showed enhanced quantitative bone regeneration; however, this was only found to be significant in the scaffold/SSCs group (p = 0.04) and complete defect bridging was not achieved in any group. The mechanical strength was significantly less than that of contralateral control tibiae (p < 0.01) and would not be appropriate for full functional loading in a clinical setting. This study explored the hypothesis that cell therapy would enhance bone formation in a critical-sized defect compared to scaffold alone, using an external fixation construct, to bridge the scale-up gap between small animal studies and potential clinical translation. The model has proved a successful critical defect and analytical techniques have been found to be both valid and reproducible. Further work is required with both scaffold production techniques and cellular protocols in order to successfully scale-up this stem cell/binary blend polymer scaffold. © 2015 The Authors. Journal of Tissue Engineering and Regenerative Medicine published by John Wiley & Sons, Ltd.

  • Role of cell-matrix interactions on VIC phenotype and tissue deposition in 3D PEG hydrogels.
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2013-10-17
    Sarah T Gould,Kristi S Anseth

    Valvular interstitial cells (VICs) respond to 3D matrix interactions in a complex manner, but understanding these effects on VIC function better is important for applications ranging from valve tissue engineering to studying valve disease. Here, we encapsulated VICs in poly(ethylene glycol) (PEG) hydrogels modified with three different adhesive ligands, derived from fibronectin (RGDS), elastin (VGVAPG) and collagen-1 (P15). By day 14, VICs became significantly more elongated in RGDS-containing gels compared to VGVAPG or P15. This difference in cell morphology appeared to correlate with global matrix metalloproteinase (MMP) activity, as VICs encapsulated in RGDS-functionalized hydrogels secreted higher levels of active MMP at day 2. VIC activation to a myofibroblast phenotype was also characterized by staining for α-smooth muscle actin (αSMA) at day 14. The percentage of αSMA+ VICs in the VGVAPG gels was the highest (56%) compared to RGDS (33%) or P15 (38%) gels. Matrix deposition and composition were also characterized at days 14 and 42 and found to depend on the initial hydrogel composition. All gel formulations had similar levels of collagen, elastin and chondroitin sulphate deposited as the porcine aortic valve. However, the composition of collagen deposited by VICs in VGVAPG-functionalized gels had a significantly higher collagen-X:collagen-1 ratio, which is associated with stenotic valves. Taken together, these data suggest that peptide-functionalized PEG hydrogels are a useful system for culturing VICs three-dimensionally and, with the ability to systematically alter biochemical and biophysical properties, this platform may prove useful in manipulating VIC function for valve regeneration. Copyright © 2013 John Wiley & Sons, Ltd.

  • Iron dose-dependent differentiation and enucleation of human erythroblasts in serum-free medium.
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2013-04-23
    Colleen Byrnes,Y Terry Lee,Emily R Meier,Antoinette Rabel,David B Sacks,Jeffery L Miller

    Improvements in ex vivo generation of enucleated red blood cells are being sought for erythroid biology research, toward the ultimate goal of erythrocyte engineering for clinical use. Based upon the high levels of iron-saturated transferrin in plasma serum, it was hypothesized that terminal differentiation in serum-free media may be highly dependent on the concentration of iron. Here adult human CD34(+) cells were cultured in a serum-free medium containing dosed levels of iron-saturated transferrin (holo-Tf, 0.1-1.0 mg/ml). Iron in the culture medium was reduced, but not depleted, with erythroblast differentiation into haemoglobinized cells. At the lowest holo-Tf dose (0.1 mg/ml), terminal differentiation was significantly reduced and the majority of the cells underwent apoptotic death. Cell survival, differentiation and enucleation were enhanced as the holo-Tf dose increased. These data suggest that adequate holo-Tf dosing is critical for terminal differentiation and enucleation of human erythroblasts generated ex vivo in serum-free culture conditions. Published 2013. This article is a US Government work and is in the public domain in the USA.

  • Mesenchymal progenitor cells derived from traumatized muscle enhance neurite growth.
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2012-05-04
    Wesley M Jackson,Peter G Alexander,Jamie D Bulken-Hoover,Jared A Vogler,Youngmi Ji,Patricia McKay,Leon J Nesti,Rocky S Tuan

    The success of peripheral nerve regeneration is governed by the rate and quality of axon bridging and myelination that occurs across the damaged region. Neurite growth and the migration of Schwann cells is regulated by neurotrophic factors produced as the nerve regenerates, and these processes can be enhanced by mesenchymal stem cells (MSCs), which also produce neurotrophic factors and other factors that improve functional tissue regeneration. Our laboratory has recently identified a population of mesenchymal progenitor cells (MPCs) that can be harvested from traumatized muscle tissue debrided and collected during orthopaedic reconstructive surgery. The objective of this study was to determine whether the traumatized muscle-derived MPCs exhibit neurotrophic function equivalent to that of bone marrow-derived MSCs. Similar gene- and protein-level expression of specific neurotrophic factors was observed for both cell types, and we localized neurogenic intracellular cell markers (brain-derived neurotrophic factor and nestin) to a subpopulation of both MPCs and MSCs. Furthermore, we demonstrated that the MPC-secreted factors were sufficient to enhance in vitro axon growth and cell migration in a chick embryonic dorsal root ganglia (DRG) model. Finally, DRGs in co-culture with the MPCs appeared to increase their neurotrophic function via soluble factor communication. Our findings suggest that the neurotrophic function of traumatized muscle-derived MPCs is substantially equivalent to that of the well-characterized population of bone marrow-derived MPCs, and suggest that the MPCs may be further developed as a cellular therapy to promote peripheral nerve regeneration.

  • In vivo formation of bone and haematopoietic territories by transplanted human bone marrow stromal cells generated in medium with and without osteogenic supplements.
    J. Tissue Eng. Regen. Med. (IF 3.319) Pub Date : 2011-11-05
    Sergei A Kuznetsov,Mahesh H Mankani,Pamela Gehron Robey

    Autologous transplantation of human bone marrow stromal cells (BMSCs) has been successfully used for bone reconstruction. However, in order to advance this approach into the mainstream of bone tissue engineering, the conditions for BMSC cultivation and transplantation must be optimized. In a recent report, cultivation with dexamethasone (Dex) significantly increased bone formation by human BMSCs in vivo. Based on this important conclusion, we analysed the data accumulated by our laboratory, where human BMSCs have been routinely generated using media both with and without a combination of two osteogenic supplements: Dex at 10(-8)  m and ascorbic acid phosphate (AscP) at 10(-4)  m. Our data demonstrate that for 22/24 donors, BMSC strains propagated with and without Dex/AscP formed similar amounts of bone in vivo. Thus, human BMSCs do not appear to need to be induced to osteogenic differentiation ex vivo prior to transplantation. Similarly, for 12/14 donors, BMSC strains cultured with and without Dex/AscP formed haematopoietic territories to a comparable extent. While Dex/AscP did not increase bone formation, they significantly stimulated BMSC in vitro proliferation without affecting the number of BMSC colonies formed by the colony-forming units-fibroblasts. We conclude that for the substantial majority of donors, Dex/AscP have no effect on the ability of BMSCs to form bone and myelosupportive stroma in vivo. However, due to increased BMSC proliferation, the total osteogenic population obtained from a single marrow sample is larger after cultivation with Dex/AscP than without them. Secondary to increased BMSC proliferation, Dex/AscP may stimulate bone formation if BMSCs and/or the transplantation system are less than optimal. Published 2011. This article is a U.S. Government work and is in the public domain in the USA.

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上海纽约大学William Glover