A Simple Dynamic Strategy to Deliver Stem Cells to Decellularized Nerve Allografts.
ABSTRACT: BACKGROUND:The addition of adipose-derived mesenchymal stromal cells to decellularized nerve allografts may improve outcomes of nerve reconstruction. Prior techniques used for cell seeding are traumatic to both the mesenchymal stromal cells and nerve graft. An adequate, reliable, and validated cell seeding technique is an essential step for evaluating the translational utility of mesenchymal stromal cell-enhanced decellularized nerve grafts. The purpose of this study was to develop a simple seeding strategy with an optimal seeding duration. METHODS:A dynamic bioreactor was used to seed rat and human mesenchymal stromal cells separately onto rat and human decellularized nerve allografts. Cell viability was evaluated by MTS assays and cellular topology after seeding was determined by scanning electron microscopy. Cell density and distribution were determined by Live/Dead assays and Hoechst staining at four different time points (6, 12, 24, and 72 hours). The validity and reliability of the seeding method were calculated. RESULTS:Cells remained viable at all time points, and mesenchymal stromal cells exhibited exponential growth in the first 12 hours of seeding. Seeding efficiency increased significantly from 79.5 percent at 6 hours to 89.2 percent after 12 hours of seeding (p = 0.004). Both intrarater reliability (r = 0.97) and interrater reliability (r = 0.92) of the technique were high. CONCLUSIONS:This study describes and validates a new method of effectively seeding decellularized nerve allografts with mesenchymal stromal cells. This method is reproducible, distributes cells homogenously over the graft, and does not traumatize the intraneural architecture of the allograft. Use of this validated seeding technique will permit critical comparison of graft outcomes.
Project description:The purpose of this study was to evaluate the molecular mechanisms underlying nerve repair by a decellularized nerve allograft seeded with adipose-derived mesenchymal stromal cells (MSCs) and compare it to the unseeded allograft and autograft nerve. Methods:Undifferentiated MSCs were seeded onto decellularized nerve allografts and used to reconstruct a 10 mm gap in a rat sciatic nerve model. Gene expression profiles of genes essential for nerve regeneration and immunohistochemical staining (IHC) for PGP9.5, NGF, RECA-1, and S100 were obtained 2 weeks postoperatively. Results:Semi-quantitative RT-PCR analysis showed that the angiogenic molecule VEGFA was significantly increased in seeded allografts, and transcription factor SOX2 was downregulated in seeded allografts. Seeded grafts showed a significant increase in immunohistochemical markers NGF and RECA-1, when compared with unseeded allografts. Conclusions:MSCs contributed to the secretion of trophic factors. A beneficial effect of the MSCs on angiogenesis was found when compared with the unseeded nerve allograft, but implanted MSCs did not show evidence of differentiation into Schwann cell-like cells.
Project description:OBJECTIVE:We aimed to investigate the functionality of human decellularized stromal laminas seeded with cultured human corneal endothelial cells as a tissue engineered endothelial graft (TEEK) construct to perform endothelial keratoplasty in an animal model of corneal endothelial damage. METHODS:Engineered corneal endothelial grafts were constructed by seeding cultured human corneal endothelial cell (hCEC) suspensions onto decellularized human corneal stromal laminas with various coatings. The functionality and survival of these grafts with cultured hCECs was examined in a rabbit model of corneal endothelial damage after central descemetorhexis. Rabbits received laminas with and without hCECs (TEEK and control group, respectively). RESULTS:hCEC seeding over fibronectin-coated laminas provided an optimal and consistent endothelial cell count density and polygonal shape on the decellularized laminas, showing active pump fuction. Surgery was performed uneventfully as standard Descemet stripping automated endothelial keratoplasty (DSAEK). Corneal transparency gradually recovered in the TEEK group, whereas haze and edema persisted for up to 4 weeks in the controls. Histologic examination showed endothelial cells of human origin covering the posterior surface of the graft in the TEEK group. CONCLUSIONS:Grafting of decellularized stroma carriers re-surfaced with human corneal endothelial cells ex vivo can be a readily translatable method to improve visual quality in corneal endothelial diseases.
Project description:Emulating autograft healing within the context of decellularized bone allografts has immediate clinical applications in the treatment of critical-sized bone defects. The periosteum, a thin, osteogenic tissue that surrounds bone, houses a heterogenous population of stem cells and osteoprogenitors. There is evidence that periosteum-cell derived paracrine factors, specifically vascular endothelial growth factor (VEGF) and bone morphogenetic protein 2 (BMP2), orchestrate autograft healing through host cell recruitment and subsequent tissue elaboration. In previous work, we demonstrated that the use of poly(ethylene glycol) (PEG) hydrogels as a tissue engineered (T.E.) periosteum to localize mesenchymal stem cells (MSCs) to the surface of decellularized bone enhances allograft healing and integration. Herein, we utilize a mixed population of 50:50 MSCs and osteoprogenitor cells to better mimic native periosteum cell population and paracrine factor production to further promote allograft healing. This mixed cell population was localized to the surface of decellularized allografts within degradable hydrogels and shown to expedite allograft healing. Specifically, bone callus formation and biomechanical graft-host integration are increased as compared to unmodified allografts. These results demonstrate the dual importance of periosteum-mediated paracrine factors orchestrating host cell recruitment as well as new bone formation while developing clinically translatable strategies for allograft healing and integration.
Project description:Nerve conduits have become an established option for repair of sensory deficits of up to 2 cm. More recently, decellularized nerve allograft has also been advocated as an option for nerve repair; however, no clinical studies have examined its efficacy for the treatment of sensory nerve defects. The aim of this study was to examine our early experience with the use of decellularized nerve allograft for repair of segmental nerve defects within the hand and fingers. From July 2007 to March 2008, seven patients who had ten nerve gaps were treated surgically using decellularized nerve allograft. Eight digital and two dorsal sensory nerves were repaired. The etiologies of the nerve defects were traumatic nerve transection in eight defects and neuroma resection and reconstruction in two defects. All of the affected nerves were pure sensory fibers. Functional recovery was evaluated by blinded hand therapist using moving and static two point discrimination tests. Implantation sites were also evaluated for any signs of infection, rejection, or graft extrusion. There were five men and two women with a mean age of 44 years (range 23-65). Mean nerve graft length was 2.23 cm with a range of 0.5-3 cm. Mean follow up time was 9 months (range 5-12). Average two point discrimination was 4.4 mm moving and 5.5 mm static at last recorded follow-up. There were no wound infections observed around the graft material and sensory improvement was observed in all of the patients despite this short-term follow-up. Re-exploration of two fingers was required for flexor tendon rupture in one and flexor tendon tenolysis in the other. In both cases, the nerve allograft was visualized and appeared well incorporated in the repair site. Decellularized nerve allografts were capable of returning adequate sensation in nerve defects ranging from 0.5 to 3 cm. There were no cases of infection or rejection. Decellularized nerve allograft may provide an option for segmental nerve gaps beyond 2 cm. Randomized comparative studies will be required to determine efficacy in comparison to collagen conduits or nerve autograft.
Project description:Structural bone allografts are widely used in the clinic to treat critical sized bone defects, despite lacking the osteoinductive characteristics of live autografts. To address this, we generated revitalized structural allografts wrapped with mesenchymal stem/progenitor cell (MSC) sheets, which were produced by expanding primary syngenic bone marrow derived cells on temperature-responsive plates, as a tissue-engineered periosteum. In vitro assays demonstrated maintenance of the MSC phenotype in the sheets, suggesting that short-term culturing of MSC sheets is not detrimental. To test their efficacy in vivo, allografts wrapped with MSC sheets were transplanted into 4-mm murine femoral defects and compared to allografts with direct seeding of MSCs and allografts without cells. Evaluations consisted of X-ray plain radiography, 3D microCT, histology, and biomechanical testing at 4- and 6-weeks post-surgery. Our findings demonstrate that MSC sheets induce prolonged cartilage formation at the graft-host junction and enhanced bone callus formation, as well as graft-host osteointegration. Moreover, a large periosteal callus was observed spanning the allografts with MSC sheets, which partially mimics live autograft healing. Finally, biomechanical testing showed a significant increase in the structural and functional properties of MSC sheet grafted femurs. Taken together, MSC sheets exhibit enhanced osteogenicity during critical sized bone defect repair, demonstrating the feasibility of this tissue engineering solution for massive allograft healing.
Project description:Chemically extracted acellular nerve allografts loaded with brain-derived neurotrophic factor-transfected or ciliary neurotrophic factor-transfected bone marrow mesenchymal stem cells have been shown to repair sciatic nerve injury better than chemically extracted acellular nerve allografts alone, or chemically extracted acellular nerve allografts loaded with bone marrow mesenchymal stem cells. We hypothesized that these allografts compounded with both brain-derived neurotrophic factor- and ciliary neurotrophic factor-transfected bone marrow mesenchymal stem cells may demonstrate even better effects in the repair of peripheral nerve injury. We cultured bone marrow mesenchymal stem cells expressing brain-derived neurotrophic factor and/or ciliary neurotrophic factor and used them to treat sciatic nerve injury in rats. We observed an increase in sciatic functional index, triceps wet weight recovery rate, myelin thickness, number of myelinated nerve fibers, amplitude of motor-evoked potentials and nerve conduction velocity, and a shortened latency of motor-evoked potentials when allografts loaded with both neurotrophic factors were used, compared with allografts loaded with just one factor. Thus, the combination of both brain-derived neurotrophic factor and ciliary neurotrophic factor-transfected bone marrow mesenchymal stem cells can greatly improve nerve injury.
Project description:OBJECTIVE:Tissue-engineered vascular grafts containing adipose-derived mesenchymal stem cells offer an alternative to small-diameter vascular grafts currently used in cardiac and lower-extremity revascularization procedures. Adipose-derived, mesenchymal stem cell-infused, tissue-engineered vascular grafts have been shown to promote remodeling and vascular homeostasis in vivo and offer a possible treatment solution for those with cardiovascular disease. Unfortunately, the time needed to cultivate adipose-derived mesenchymal stem cells remains a large hurdle for tissue-engineered vascular grafts as a treatment option. The purpose of this study was to determine if stromal vascular fraction (known to contain progenitor cells) seeded tissue-engineered vascular grafts would remain patent in vivo and remodel, allowing for a "same-day" process for tissue-engineered vascular graft fabrication and implantation. METHODS:Stromal vascular fraction, obtained from adult human adipose tissue, was seeded within 4 hours after acquisition from the patient onto poly(ester urethane)urea bilayered scaffolds using a customized rotational vacuum seeding device. Constructs were then surgically implanted as abdominal aortic interposition grafts in Lewis rats. RESULTS:Findings revealed patency in 5 of 7 implanted scaffolds at 8 weeks, along with neotissue formation and remodeling occurring in patent tissue-engineered vascular grafts. Patency was documented using angiography and gross inspection, and remodeling and vascular components were detected using immunofluorescent chemistry. CONCLUSIONS:A "same-day" cell-seeded, tissue-engineered vascular graft can remain patent after implantation in vivo, with neotissue formation and remodeling occurring by 8 weeks.
Project description:Peripheral nerve gap injuries continue to present a clinical challenge to today's surgeons. One method of surgical repair, implantation of acellular allografts, has been developed with the aim of bridging the gap with a cadaveric graft after removal of its cellular components, thereby accelerating axonal regeneration and eliminating the need for immunosuppression in recipient patients. Although decellularizing allografts reduces rates of graft rejection, the same chemical processing modifies the neural microenvironment, removing neutrotrophic factors and modifying the complex extracellular matrix. In this study, we explore 3 common methods for producing acellular allografts. Extracellular matrix content remaining after processing was investigated and was found to be highly dependent on the decellularization method. In addition, scanning electron micrographs were obtained to evaluate the structural effects of the decellularization methods. Though the content and structure of these processed allografts will contribute to their effectiveness as nerve gap repair candidates, we demonstrate that it also affects their capacity to be supplemented/preloaded with the prototypical neurotrophin, nerve growth factor (NGF), essential to neuronal regeneration. Although all allografts had some capacity for retaining NGF in the first 24 hours, only Sondell-processed grafts retained NGF over the entire experimental period of 21 days. Future studies will include validating these processed and supplemented allografts as viable alternatives to traditional autograft nerve gap repair.
Project description:Allogeneic lung transplant is limited both by the shortage of available donor lungs and by the lack of suitable long-term lung assist devices to bridge patients to lung transplantation. Avian lungs have different structure and mechanics resulting in more efficient gas exchange than mammalian lungs. Decellularized avian lungs, recellularized with human lung cells, could therefore provide a powerful novel gas exchange unit for potential use in pulmonary therapeutics. To initially assess this in both small and large avian lung models, chicken (Gallus gallus domesticus) and emu (Dromaius novaehollandiae) lungs were decellularized using modifications of a detergent-based protocol, previously utilized with mammalian lungs. Light and electron microscopy, vascular and airway resistance, quantitation and gel analyses of residual DNA, and immunohistochemical and mass spectrometric analyses of remaining extracellular matrix (ECM) proteins demonstrated maintenance of lung structure, minimal residual DNA, and retention of major ECM proteins in the decellularized scaffolds. Seeding with human bronchial epithelial cells, human pulmonary vascular endothelial cells, human mesenchymal stromal cells, and human lung fibroblasts demonstrated initial cell attachment on decellularized avian lungs and growth over a 7-day period. These initial studies demonstrate that decellularized avian lungs may be a feasible approach for generating functional lung tissue for clinical therapeutics.
Project description:Decellularized natural bladder matrices (neobladders) represent an exciting means to regenerate the bladder following bladder cancer-associated cystectomy. In this study, we compare the evolution of decellularized matrices with recellularized matrices by seeding it with human adipose-derived mesenchymal stem cells (ADSC) after implantation following partial cystectomy in rats. We discovered significant anatomical differences since 10 days after neobladder implantation with the ADSC-containing matrices promoting a significant recovery of mature p63- and cytokeratin 7-positive urothelium. We also discovered significantly induced expression of the vimentin mesoderm marker in the submucosal layer in ADSC-seeded matrices. Interestingly, we found a higher expression of smooth muscle actin in transversal and longitudinal smooth muscle layers with ADSC-seeded matrices. Furthermore, ADSC also showed increased vascularization and nerve innervation of the neobladder as determined by the distribution of CD31 and S100? reactivity, respectively. We believe that ADSC and their paracrine-acting pro-regenerative secretome within decellularized matrices represent an efficient bladder substitution strategy; however, we require a fuller understanding of the mechanisms involved before clinical studies can begin.