Cell-seeded Scaffolds Research Articles

Effect of ‘in air’ freezing on post-thaw recovery of Callithrix jacchus mesenchymal stromal cells and properties of 3D collagen-hydroxyapatite scaffolds

Through enabling an efficient supply of cells and tissues in the health sector on demand, cryopreservation is increasingly becoming one of the mainstream technologies in rapid translation and commercialization of regenerative medicine research. Cryopreservation of tissue-engineered constructs (TECs) is an emerging trend that requires the development of practically competitive biobanking technologies. In our previous studies, we demonstrated that conventional slow-freezing using dimethyl sulfoxide (Me2SO) does not provide sufficient protection of mesenchymal stromal cells (MSCs) frozen in 3D collagen-hydroxyapatite scaffolds. After simple modifications to a cryopreservation protocol, we report on significantly improved cryopreservation of TECs.Porous 3D scaffolds were fabricated using freeze-drying of a mineralized collagen suspension and following chemical crosslinking. Amnion-derived MSCs from common marmoset monkey Callithrix jacchus were seeded onto scaffolds in static conditions. Cell-seeded scaffolds were subjected to 24 h pre-treatment with 100 mM sucrose and slow freezing in 10% Me2SO/20% FBS alone or supplemented with 300 mM sucrose. Scaffolds were frozen ‘in air’ and thawed using a two-step procedure. Diverse analytical methods were used for the interpretation of cryopreservation outcome for both cell-seeded and cell-free scaffolds. In both groups, cells exhibited their typical shape and well-preserved cell-cell and cell-matrix contacts after thawing. Moreover, viability test 24 h post-thaw demonstrated that application of sucrose in the cryoprotective solution preserves a significantly greater portion of sucrose-pretreated cells (more than 80%) in comparison to Me2SO alone (60%).No differences in overall protein structure and porosity of frozen scaffolds were revealed whereas their compressive stress was lower than in the control group. In conclusion, this approach holds promise for the cryopreservation of ‘ready-to-use’ TECs.

The application of dried amniotic membrane scaffold with adipose derived-mesenchymal stem cell seeding as graft in urethral reconstruction (experiment on rabbit)

BackgroundUrethral reconstruction has been a problem for a long time and is a major challenge for surgeons. Surgical management of urethral defects is still controversial because there is no synthetic material that is considered ideal as a substitute for the urethra. With the development of adipose-derived mesenchymal stem cells, this research aims to observe the difference between the use of dried amniotic membrane as a scaffold with and without mesenchymal stem cells seeding as graft in urethral reconstruction. MethodsThis research was a pure experimental research with a post-test only control group design using 24 male New Zealand white rabbits. The samples were divided into 3 groups: one control group, one scaffold (dried amniotic membrane) without stem cell seeding group, and one stem cell-seeded scaffold group. The rabbits were evaluated at 28 days after treatment and the results were analyzed using Chi-Square test. ResultsThe number of urethras that were healed were higher in stem cell-seeded scaffold compared to scaffold without stem cell seeding group (p < 0.05). ConclusionConsidering the promising result, application of dried amniotic membrane as a scaffold with adipose-derived mesenchymal stem cell seeding could be an option for urethral reconstruction.

Fractional CO2 laser micropatterning of cell-seeded electrospun collagen scaffolds enables rete ridge formation in 3D engineered skin

Rete ridges are interdigitations of the epidermis and dermis of the skin that play multiple roles in homeostasis, including enhancing adhesion via increased contact area and acting as niches for epidermal stem cells. These structures, however, are generally absent from engineered skin (ES). To develop ES with rete ridges, human fibroblast-seeded dermal templates were treated with a fractional CO2 laser, creating consistently spaced wells at the surface. Constructs with and without laser treatment were seeded with keratinocytes, cultured for 10 days, and grafted onto athymic mice for four weeks. Rete-ridge like structures were observed in the laser-patterned (ridged) samples at the time of grafting and were maintained in vivo. Ridged grafts displayed improved barrier function over non-lasered (flat) grafts at the time of grafting and 4 weeks post-grafting. Presence of ridges in vivo corresponded with increased keratinocyte proliferation, epidermal area, and basement membrane length. These results suggest that this method can be utilized to develop engineered skin grafts with rete ridges, that the ridge pattern is stable for at least 4 weeks post-grafting, and that the presence of these ridges enhances epidermal proliferation and establishment of barrier function. Statement of significanceRete ridges play a role in epidermal homeostasis, enhance epidermal-dermal adhesion and act as niches for epidermal stem cells. Despite their role in skin function, these structures are not directly engineered into synthetic skin. A new method to rapidly and reproducibly generate rete ridges in engineered skin was developed using fractional CO2 laser ablation. The resulting engineered rete ridges aided in the establishment of epidermal barrier function, basement membrane protein deposition and epidermal regeneration. This new model of engineered skin with rete ridges could be utilized as an in vitro system to study epidermal stem cells, a testbed for pharmaceutical evaluation or translated for clinical use in full-thickness wound repair.

Improving cell distribution on 3D additive manufactured scaffolds through engineered seeding media density and viscosity

In order to ensure the long-term in vitro and in vivo functionality of cell-seeded 3D scaffolds, an effective and reliable method to control cell seeding efficiency and distribution is crucial. Static seeding on 3D additive manufactured scaffolds made of synthetic polymers still remains challenging, as it often results in poor cell attachment, high cell sedimentation and non-uniform cell distribution, due to gravity and to the intrinsic macroporosity and surface chemical properties of the scaffolds. In this study, the biocompatible macromolecules dextran and Ficoll (Ficoll-Paque) were used for the first time as temporary supplements to alter the viscosity and density of the seeding media, respectively, and improve the static seeding output. The addition of these macromolecules drastically reduced the cell sedimentation velocities, allowing for homogeneous cell attachment to the scaffold filaments. Both dextran and Ficoll-Paque -based seeding methods supported human mesenchymal stromal cells viability and osteogenic differentiation post-seeding. Interestingly, the improved cell distribution led to increased matrix production and mineralization compared to scaffolds seeded by conventional static method. These results suggest a simple and universal method for an efficient seeding of 3D additive manufactured scaffolds, independent of their material and geometrical properties, and applicable for bone and various other tissue regeneration. Statement of significanceAdditive manufacturing has emerged as one of the desired technologies to fabricate complex and patient-specific 3D scaffolds for bone regeneration. Along with the technology, new synthetic polymeric materials have been developed to meet processability requirements, as well as the mechanical properties and biocompatibility necessary for the application. Yet, there is still lack of methodology for a universal cell seeding method applicable to all additive manufactured 3D scaffolds regardless of their characteristics. We believe that our simple and reliable method, which is based on adjusting the cell settling velocity to aid cell attachment, could potentially help to maximize the efficiency, and therefore, functionality of cell-seeded constructs. This is of great importance when aiming for both in vitro and future clinical applications.

Towards clinical application of tissue engineering for erectile penile regeneration.

Penile wounds after traumatic and surgical amputation require reconstruction in the form of autologous tissue transfers. However, currently used techniques are associated with high infection rates, implant erosion and donor site morbidity. The use of tissue-engineered neocorpora provides an alternative treatment option. Contemporary tissue-engineering strategies enable the seeding of a biomaterial scaffold and subsequent implantation to construct a neocorpus. Tissue engineering of penile tissue should focus on two main strategies: first, correcting the volume deficit for structural integrity in order to enable urinary voiding in the standing position and second, achieving erectile function for sexual activity. The functional outcomes of the neocorpus can be addressed by optimizing the use of stem cells and scaffolds, or alternatively, the use of gene therapy. Current research in penile tissue engineering is largely restricted to rodent and rabbit models, but the use of larger animal models should be considered as a better representation of the anatomical and physiological function in humans. The development of a cell-seeded scaffold to achieve and maintain erection continues to be a considerable challenge in humans. However, advances in penile tissue engineering show great promise and, in combination with gene therapy and surgical techniques, have the potential to substantially improve patient outcomes.

Osteogenic potential of induced pluripotent stem cells from human adipose-derived stem cells

BackgroundBone regeneration is a crucial and challenging issue in clinical practice. Bone tissue engineering (BTE) with an optimal cell source may provide an ideal strategy for the reconstruction of bone defects. This study examined whether induced pluripotent stem cells (iPSCs) derived from adipose-derived stem cells (ASCs) could act as an osteogenic substitute and whether these ASC-iPSCs yield more new bone formation than ASCs in hydrogel scaffolds.MethodsASC-iPSCs were reprogrammed from ASCs through a retroviral system. ASCs were harvested and isolated from adipose tissue of humans. An aliquot of cell suspension (1 × 106 cells/mL) was seeded directly onto the nHAP-gelatin cryogel scaffolds. Nude mice back implantation of cell-seeded scaffolds was designed for in vivo comparison of osteogenic potentials between ASCs and ASC-iPSCs. Samples were harvested 4 and 8 weeks after implantation for further analysis based on histology and RT-PCR.ResultsASC-iPSCs were successfully obtained from human adipose-derived stem cells. PCR results also showed that specific genes of iPSCs with the ability to cause the differentiation of cells into the three germ layers were expressed. In our in vivo experiments, iPSCs were subcutaneously injected into nude mice to induce teratoma formation. The morphology of the three germ layers was confirmed by histological staining. ASC is an essential cell source for BTE with benefits of high volume and less-invasive acquisition. With additional transforming Yamanaka factors, ASC-iPSCs showed higher osteogenic differentiation and elevated expression of collagen type I (Col I), osteocalcin (OCN), alkaline phosphate (ALP), and runt-related transcription factor 2 (RunX-2).ConclusionsThis report suggests that ASC-iPSCs could be a superior cell source in BTE with better osteogenic differentiation efficacy for future clinical applications.

Core-Shell Nanofibrous Scaffolds for Repair of Meniscus Tears.

Electrospinning is an attractive method of fabricating nanofibers that replicate the ultrastructure of the human meniscus. However, it is challenging to approximate the mechanical properties of meniscal tissue while maintaining the biocompatibility of collagen fibers. Our objective was to determine if functionalizing polylactic acid (PLA) nanofibers with collagen would enhance their biocompatibility. We therefore used coaxial electrospinning to generate core-shell nanofibers with a core of PLA for mechanical strength and a shell of collagen to enhance cell attachment and matrix synthesis. We characterized the nanostructure of the engineered scaffolds and measured the hydrophilic and mechanical properties. We assessed the performance of human meniscal cells seeded on coaxial electrospun scaffolds to produce meniscal tissue by gene expression and histology. Finally, we investigated whether these cell-seeded scaffolds could repair surgical tears created ex vivo in avascular meniscal explants. Histology, immunohistochemistry, and mechanical testing of ex vivo repair provided evidence of neotissue that was significantly better integrated with the native tissue than with the acellular coaxial electrospun scaffolds. Human meniscal cell-seeded coaxial electrospun scaffolds may have potential in enhancing repair of avascular meniscus tears. Impact Statement The success of any tissue-engineered meniscus graft relies on its ability to mimic native three-dimensional microstructure, support cell growth, produce tissue-specific matrix, and enhance graft integration into the repair site. Polylactic acid scaffolds possess the desired mechanical properties, whereas collagen scaffolds induce better cell attachment and enhanced tissue regeneration. We therefore fabricated nanofibrous scaffolds that combined the properties of two biomaterials. These novel coaxial scaffolds more closely emulated the structure, mechanical properties, and biochemical composition of native meniscal tissue. Our findings of meniscogenic tissue generation and integration in meniscus defects have the potential to be translated to clinical use.

Bone allografts combined with adipose-derived stem cells in an optimized cell/volume ratio showed enhanced osteogenesis and angiogenesis in a murine femur defect model

Critical sized defects, especially in long bones, pose one of the biggest problems in orthopedic surgery. By definition, these defects do not heal without further treatment. Different therapeutic options range from autologous bone grafts, for example, free vascularized bone grafts, to commercially available bone allografts. Disadvantages of these bone allografts are related to reduced osteogenesis, since they are solely composed of cell-free bone matrix. The purpose of this study was to investigate the cell seeding efficiency of human adipose-derived stem cells (hASCs) on human bone allografts in vitro and furthermore analyze these optimized seeded allografts in a critical sized defect model in vivo. Cancellous human bone allografts were colonized with human ASCs in vitro. Cell seeding efficiency was evaluated by Cell Counting Kit-8 assay. Thereafter, optimized hASC-seeded bone scaffolds were examined in a murine femur defect model, stabilized with the MouseExFix system. Subsequently, x-ray analysis and histology were performed. Examination of cell seeding efficiency revealed an optimum starting population of 84,600 cells per 100mm3 scaffold. In addition, scaffolds seeded with hASCs showed increased osteogenesis compared with controls. Histological analysis revealed increased remodeling and elevated new bone formation within hASC-seeded scaffolds. Moreover, immunohistochemical stainings revealed increased proliferation, osteogenesis, and angiogenesis. In this study, we systemically optimized cell/volume ratio of two promising components of tissue engineering: hASCs and human bone allografts. These findings may serve as a basis for future translational studies. KEY MESSAGES: Bone tissue engineering. Mesenchymal stem cells derived from human adipose tissue (hASCs). Optimal cell/volume ratio of cell-seeded scaffolds. Increased osteogenesis and angiogenesis in vivo.

Hybrid nanocomposite as a chest wall graft with improved integration by adipose-derived stem cells

Surgery of the chest wall is potentially required to cover large defects after removal of malignant tumours. Usually, inert and non-degradable Gore-Tex serves to replace the missing tissue. However, novel biodegradable materials combined with stem cells are available that stimulate the healing. Based on poly-lactic-co-glycolic acid and amorphous calcium phosphate nanoparticles (PLGA/aCaP) and pure PLGA, a dual layer biodegradable hybrid nanocomposite was generated. Mouse adipose-derived stem cells were cultered on electrospun disks (ASCs of C57BL/6), and biomechanical tests were performed. The cell-seeded scaffolds were engrafted in C57BL/LY5.1 mice to serve as a chest wall substitute. Cell invasion into the bi-layered material, extent of CD45+ cells, inflammatory response, neo-vascularization and ECM composition were determined at 1 and 2 months post-surgery, respectively. The bi-layered hybrid nanocomposite was stable after a 2-week in vitro culture, in contrast to PLGA/aCaP without a PLGA layer. There was a complete biointegration and good vascularization in vivo. The presence of ASCs attracted more CD45+ cells (hematopoietic origin) compared to cell-free scaffolds. Inflammatory reaction was similar for both groups (±ASCs) at 8 weeks. A bi-layered hybrid nanocomposite fabricated of electrospun PLGA/aCaP and a reinforcing layer of pristine PLGA is an ideal scaffold for chest wall reconstruction. It is stable and allows a proper host tissue integration. If ASCs are seeded, they attract more CD45+ cells, supporting the regeneration process.

Tissue Constructs with Human Adipose-Derived Mesenchymal Stem Cells to Treat Bone Defects in Rats.

The use of porous scaffolds created by additive manufacturing is considered a viable approach for the regeneration of critical-size bone defects. This paper investigates the xenotransplantation of polycaprolactone (PCL) tissue constructs seeded with differentiated and undifferentiated human adipose-derived mesenchymal stem cells (hADSCs) to treat calvarial critical-sized defect in Wistar rats. PCL scaffolds without cells were also considered. In vitro and in vivo biological evaluations were performed to assess the feasibility of these different approaches. In the case of cell seeded scaffolds, it was possible to observe the presence of hADSCs in the rat tissue contributing directly (osteoblasts) and indirectly (stimulation by paracrine factors) to tissue formation, organization and mineralization. The presence of bone morphogenetic protein-2 (BMP-2) in the rat tissue treated with cell-seeded PCL scaffolds suggests that the paracrine factors of undifferentiated hADSC cells could stimulate BMP-2 production by surrounding cells, leading to osteogenesis. Moreover, BMP-2 acts synergistically with growth factors to induce angiogenesis, leading to higher numbers of blood vessels in the groups containing undifferentiated and differentiated hADSCs.

Review of clinical experience on biomaterials and tissue engineering of urinary bladder.

In recent pre-clinical studies, biomaterials and bladder tissue engineering have shown promising outcomes when addressing the need for bladder tissue replacement. To date, multiple clinical experiences have been reported. Herein, we aim to review and summarize the reported clinical experience of biomaterial usage and tissue engineering of the urinary bladder. A systematic literature search was performed on Feb 2019 to identify clinical reports on biomaterials for urinary bladder replacement or augmentation and clinical experiences with bladder tissue engineering. We identified and reviewed human studies using biomaterials and tissue-engineered bladder as bladder substitutes or augmentation implants. The studies were then summarized for each respective procedure indication, technique, follow-up period, outcome, and important findings of the studies. An extensive literature search identified 25 studies of case reports and case series with a cumulative clinical experience of 222 patients. Various biomaterials and tissue-engineered bladder were used, including plastic/polyethylene mold, preserved dog bladder, gelatine sponge, Japanese paper with Nobecutane, lypholized human dura, bovine pericardium, amniotic membrane, small intestinal mucosa, and bladder tissue engineering with autologous cell-seeded biodegradable scaffolds. However, overall clinical experiences including the outcomes and safety reports were not satisfactory enough to replace enterocystoplasty. To date, several clinical experiences of biomaterials and tissue-engineered bladder have been reported; however, various studies have reported non-satisfactory outcomes. Further technological advancements and a better understanding is needed to advance bladder tissue engineering as a future promising management option for patients requiring bladder drainage.

Tissue-Engineered Esophagus via Bioreactor Cultivation for Circumferential Esophageal Reconstruction.

The use of biomaterials for circumferential esophageal repair is technically challenging in a rat model, and an optimal scaffold implantation technique with nutritional support is essential. The purpose of this study was to investigate the effects of three-dimensional printed esophageal grafts and bioreactor cultivation on muscle regeneration and reepithelialization from circumferential esophageal defects in a rat model. Here, we designed an artificial esophagus that can enhance the regeneration of esophageal mucosa and muscle through the optimal combination of a two-layered tubular scaffold and mesenchymal stem cell-based bioreactor system. The graft was verified by the performance comparison with an omentum-cultured esophageal scaffold. We also applied a new surgical anastomosis technique and a thyroid gland flap over the implanted scaffold to improve graft survival. Although no regenerated mucosal layer was observed around the implants of the control group, histological examination of the regenerative esophagi along the scaffold revealed that the bioreactor system and omentum-cultured groups showed more than 80% of the mucosal regeneration without a fistula. The regenerated tissues showed that the integration of the esophageal scaffold and its native esophageal tissue was intact and were covered with layers of stratified squamous epithelium with several newly developed blood vessels. Therefore, this study describes a novel approach for circumferential esophageal reconstruction. Impact Statement In vivo functional esophageal reconstruction remains challenging due to anastomosis site leakage and necrosis of the implanted scaffold in a circumferential esophageal defect. Therefore, it is necessary to develop a tissue-engineered esophagus that enables regeneration of esophageal mucosa and muscle without leakage of the esophageal anastomosis. In this study, we proposed an intriguing strategy that combines a mesenchymal stem cell-seeded tubular scaffold with a bioreactor system for esophageal reconstruction and introduced a new surgical anastomosis technique with the thyroid gland flap over the implanted scaffold to improve graft survival. We believe that this system should be a powerful platform for circumferential replacement of the esophagus in a rat model.

Perfusion Bioreactor Culture of Bone Marrow Stromal Cells Enhances Cranial Defect Regeneration.

Cell-seeded biomaterial scaffolds have been proposed as a future option for reconstruction of bone tissue. The ability to generate larger, functional volumes of bone has been a challenge that may be addressed through the use of perfusion bioreactors. In this study, the authors investigated use of a tubular perfusion bioreactor system for the growth and differentiation of bone marrow stromal (mesenchymal stem) cells seeded onto fibrin, a highly angiogenic biomaterial. Cells were encapsulated within fibrin beads and cultured either within a tubular perfusion bioreactor system or statically for up to 14 days. Scaffolds were analyzed for osteogenic differentiation. A rodent cranial defect model (8-mm diameter) was used to assess the bone regeneration of scaffolds cultured in the bioreactor, statically, or used immediately after formation. Immunohistochemistry was used to visualize CD31 vessel density. Micro-computed tomographic imaging was used to visualize mineral formation within the defect volume. Tubular perfusion bioreactor system-cultured samples showed significantly greater osteodifferentiation, indicated by an increase in VEGF expression and mineral deposition, compared with statically cultured samples. Increased expression of OPN, RUNX2, VEGF, and CD90 was seen over time in both culture methods. After implantation, bioreactor samples exhibited greater bone formation and vessel density compared with all other groups. Analysis of micro-computed tomographic images showed full union formation through the greatest diameter of the defect in all bioreactor samples and the highest levels of mineralized volume after 8 weeks. Mesenchymal stem cells encapsulated in fibrin beads and cultured in the tubular perfusion bioreactor system resulted in increased vascularization and mineralized tissue formation in vivo relative to static culture.

Cell-seeded porous silk fibroin scaffolds promotes axonal regeneration and myelination in spinal cord injury rats

Accumulating evidence indicates that a suitable scaffold designed for the spinal cord injury (SCI) was needed to enhance the survival of transplanted Bone mesenchymal stem cells (BMSCs) and to promote nerve regeneration. The current study was aimed to evaluate the effect of the porous silk fibroin scaffold (PSFSs) seeded with BMSCS on nerve regeneration, myelination and functional recovery after SCI. We previously demonstrated that the PSFSs could bridge defected nerve with nerve fibers when applied to the transected spinal cord. And we found that BMSCs were adhered to the scaffold closely and have good biological compatibility with PSFSs. PSFSs seeded with BMSCs exhibited significant improvement in complete transverse thoracic SCI rat models. Flow cytometric assay also indicated that BMSCs grew well and adhered closely to the surface of the scaffold. The Basso-Beattie-Bresnehan (BBB) scores at each time point showed that the hindlimb motor function of each transplant group was also significantly restored. Meanwhile, growth associated protein 43 (GAP-43)marker of damaged axons regeneration and myelin basic protein (MBP) marker of maintaining the myelin structural and functional integrity, all markedly increased in PSFSs seeded with BMSCs models. Together, these results demonstrated that transplantation of PSFSs seeded with BMSCS could promote the nerve regeneration, myelination and functional recovery after SCI.

Bone Marrow Mesenchymal Stem Cells and Endothelial Progenitor Cells Co-Culture Enhances Large Segment Bone Defect Repair.

People have been looking for tissue engineering approaches to treat large segment bone defects as replacements of autologous bone method. Cell-seeded scaffolds are promising candidates, but lack of vascularization into the scaffolds has greatly hindered their applications. To address this problem, we used a co-culture of bone marrow derived mesenchymal stem cells (BMSCs) and endothelial progenitor cells (EPCs) on calcium phosphate ceramics scaffolds for better vascularization and thus osteogenesis. Different ratios of BMSC/EPC (3:1, 2:1, 1:1, 1:2, 1:3) in the co-culture were examined in vitro, and it was revealed that the optimal value for neovascularization and osteogenesis was 1:3 and 2:1, respectively. Then cell mixtures with the optimized ratios were cultured in distinct regions of volume-reduced scaffolds and allowed to culture for 7 days for sufficient cell adhesion and ingrowth, as demonstrated by cell proliferation throughout the scaffolds and enhanced expressions of cell markers and growth factors comparing to a mono-culture of BMSCs. Upon implantation into a rabbit large segmental bone defect model, the scaffolds with co-culture of the cells had better osteoid tissue formation and bone remodeling supported by neovascularization, comparing to scaffolds with mono-culture or without cells. Yet with sub-optimal efficacy comparing to autologous bone grafts, it was believed to be a promising candidate for treatment of large segment bone defects.

The establishment and biological assessment of a whole tissue-engineered intervertebral disc with PBST fibers and a chitosan hydrogel in vitro and in vivo.

Intervertebral disc (IVD) degeneration (IDD) is the main cause of low back pain in the clinic. In the advanced stage of IDD, both cell transplantation and gene therapy have obvious limitations. At this stage, tissue-engineered IVDs (TE-IVDs) provide new hope for the treatment of this disease. We aimed to construct a TE-IVD with a relatively complete structure. The inner annulus fibrosus (AF) was constructed using poly (butylene succinate-co-terephthalate) copolyester (PBST) electrospun fibers, and the outer AF consisted of solid PBST. The nucleus pulposus (NP) scaffold was constructed using a chitosan hydrogel, as reported in our previous research. The three components were assembled in vitro, and the mechanical properties were analyzed. AF and NP cells were implanted on the corresponding scaffolds. Then, the cell-seeded scaffolds were implanted subcutaneously in nude mice and cultured for 4 weeks; then they were removed and implanted into New Zealand white rabbits. After 4 weeks, their properties were analyzed. The PBST outer AF provided mechanical support for the whole TE-IVD. The electrospun film and chitosan hydrogel simulated the natural structure of the IVD well. Its mechanical property could meet the requirement of the normal IVD. Four weeks later, X-ray and MR imaging examination results suggested that the height of the intervertebral space was retained. The cells on the TE-IVD expressed extracellular matrix, which indicated that the cells maintained their biological function. Therefore, we conclude that the whole TE-IVD has biological and biomechanical properties to some extent, which is a promising candidate for IVD replacement therapies. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 2305-2316, 2019.

Osteointegration of a Novel Silk Fiber–Based ACL Scaffold by Formation of a Ligament-Bone Interface

Background: Given the unsatisfactory results and reported drawbacks of anterior cruciate ligament (ACL) reconstruction, such as donor site morbidity and the limited choice of grafts in revision surgery, new regenerative approaches based on tissue-engineering strategies are currently under investigation. Purposes: To determine (1) if a novel silk fiber–based ACL scaffold is able to initiate osteointegration in the femoral and tibial bone tunnels under in vivo conditions and (2) if the osteointegration process will be improved by intraoperatively seeding the scaffolds with the autologous stromal vascular fraction, an adipose-derived, stem cell–rich isolate from knee fat pads. Study Design: Controlled laboratory study. Methods: A total of 33 sheep underwent ACL resection and were then randomly assigned to 2 experimental groups: ACL reconstruction with a scaffold alone and ACL reconstruction with a cell-seeded scaffold. Half of the sheep in each group were randomly chosen and euthanized 6 months after surgery and the other half at 12 months. To analyze the integration of the silk-based scaffold in the femoral and tibial bone tunnels, hard tissue histology and micro–computed tomography measurements were performed. Results: Hard tissue histological workup showed that in all treatment groups, with or without the application of the autologous stromal vascular fraction, an interzone of collagen fibers had formed between bone and silk-based graft. This collagen-fiber continuity partly consisted of Sharpey fibers, comparable with tendon-bone healing known for autografts and allografts. Insertion sites were more broad based at 6 months and more concentrated on the slightly protruding, bony knoblike structures at 12 months. Histologically, no differences between the treatment groups were detectable. Analysis of micro–computed tomography measurements revealed a significantly higher tissue density for the cell-seeded scaffold group as compared with the scaffold-alone group in the tibial but not femoral bone tunnel after 12 months of implantation. Conclusion: The novel silk fiber–based scaffold for ACL regeneration demonstrated integration into the bone tunnels via the formation of a fibrous interzone similar to allografts and autografts. Histologically, additional cell seeding did not enhance osteointegration. No significant differences between 6 and 12 months could be detected. After 12 months, there was still a considerable amount of silk present, and a longer observation period is necessary to see if a true ligament-bone enthesis will be formed. Clinical Relevance: ACL regeneration with a silk fiber–based scaffold with and without additional cell seeding may provide an alternative treatment option to current techniques of surgical reconstruction.

A computational reaction-diffusion model for biosynthesis and linking of cartilage extracellular matrix in cell-seeded scaffolds with varying porosity.

Cartilage tissue engineering is commonly initiated by seeding cells in porous materials such as hydrogels or scaffolds. Under optimal conditions, the resulting engineered construct has the potential to fill regions where native cartilage has degraded or eroded. Within a cell-seeded scaffold supplied by nutrients and growth factors, extracellular matrix accumulation should occur concurrently with scaffold degradation. At present, the interplay between cell-mediated synthesis and linking of matrix constituents and the evolving scaffold properties is not well understood. We develop a computational model of extracellular matrix accumulation in a cell-seeded scaffold based on a continuum reaction-diffusion system with inhomogeneous inclusions representing individual cells. The effects of porosity on engineered tissue outcomes is accounted for via the use of mixture variables capturing the spatiotemporal dynamics of both bound and unbound system constituents. The unbound constituents are the nutrients and unlinked extracellular matrix, while the bound constituents are the scaffold and the linked extracellular matrix. The linking model delineates binding of matrix constituents to either existing bound extracellular matrix or to scaffold. Results on a representative domain exhibit bound matrix trapping (vs spreading) around cells in scaffolds with lower (vs higher) initial porosity, similar to experimental results obtained by Erickson et al. (Osteoarthr Cartil 17:1639-1648, 2009). Significant alterations in the spatiotemporal accumulation of bound matrix are observed when, among the set of all model parameters, only the initial scaffold porosity is varied. The model presented herein proposes a methodology to investigate coupling between cell-mediated biosynthesis and linking of extracellular matrix in porous, cell-seeded scaffolds that has the potential to aid in the design of optimal tissue-engineered cartilage constructs.

Bioengineered bladder patches constructed from multilayered adipose-derived stem cell sheets for bladder regeneration

Cell-seeded scaffolds are a common route of cell transplantation for bladder repair and reconstruction. However, when cell suspensions are harvested, proteolytic enzymes often cause extracellular matrix damage and loss of intercellular junctions. To overcome this problem, we developed a bioengineered three-dimensional bladder patch comprising porous scaffolds and multilayered adipose-derived stem cell (ASC) sheets, and evaluated its feasibility for bladder regeneration in a rat model. Adipose-derived stem cells (ASCs) were labeled with ultrasmall super-paramagnetic iron oxide (USPIO) nanoparticles. ASC patches were constructed using multilayered USPIO-labeled ASC sheets and porous polyglycolic acid scaffolds. To monitor the distribution and localization of bioengineered bladder patches in live animals, magnetic resonance imaging (MRI) was performed 2 weeks, 4 weeks and 8 weeks after transplantation. The bladder regenerative potential of ASC patches was further evaluated by urodynamic and histological analysis. Scanning electron microscopy indicated that cell sheets adhered tightly to the scaffold. MRI showed hypointense signals that lasted up to 8 weeks at the site of USPIO-labeled ASC sheet transplants. Immunofluorescence demonstrated that these tissue-engineered bladder patches promoted regeneration of urothelium, smooth muscle, neural cells and blood vessels. Urodynamic testing revealed that the ASC patch restored bladder function with augmented capacity. The USPIO-labeled ASC patch provides a promising perspective on image-guided tissue engineering and holds great promise as a safe and effective therapeutic strategy for bladder regeneration. Statement of SignificanceAdipose-derived stem cell (ASC) sheets avoid enzymatic dissociation and preserve the cell-to-cell interactions and extracellular matrix (ECM) proteins, which exhibit great potential for tissue regeneration. In this study, we developed a bioengineered three-dimensional bladder patch comprising porous scaffolds and multilayered ASC sheets, and evaluated its feasibility for bladder regeneration in a rat model. Tissue-engineered bladder patches restored bladder function and promoted regeneration of urothelium, smooth muscle, neural cells and blood vessels. Moreover, ultrasmall super-paramagnetic iron oxide (USPIO)-labeled bladder patches can be dynamically monitored in vivo by noninvasive MRI for long periods of time. Therefore, The USPIO-labeled bladder patch provides a promising image-guided therapeutic strategy for bladder regeneration.

A mouse model for the study of transplanted bone marrow mesenchymal stem cell survival and proliferation in lumbar spinal fusion.

Bone marrow aspirate has been successfully used alongside a variety of grafting materials to clinically augment spinal fusion. However, little is known about the fate of these transplanted cells. Herein, we develop a novel murine model for the in vivo monitoring of implanted bone marrow cells (BMCs) following spinal fusion. A clinical-grade scaffold was implanted into immune-intact mice undergoing spinal fusion with or without freshly isolated BMCs from either transgenic mice which constitutively express the firefly luciferase gene or syngeneic controls. The in vivo survival, distribution and proliferation of these luciferase-expressing cells was monitored via bioluminescence imaging over a period of 8weeks and confirmed via immunohistochemistry. MicroCT imaging was performed 8weeks to assess fusion. Bioluminescence imaging indicated transplanted cell survival and proliferation over the first 2weeks, followed by a decrease in cell numbers, with transplanted cell survival still evident at the end of the study. New bone formation and increased fusion mass volume were observed in mice implanted with cell-seeded scaffolds. By enabling the tracking of transplanted bone marrow-derived cells during spinal fusion in vivo, this mouse model will be integral to developing a deeper understanding of the biological processes underlying spinal fusion in future studies. These slides can be retrieved under Electronic Supplementary Material.