Stimuli-responsive peptide assemblies: Design, self-assembly, modulation, and biomedical applications.

Peptide molecules have design flexibility, self-assembly ability, high biocompatibility, good biodegradability, and easy functionalization, which promote their applications as versatile biomaterials for tissue engineering and biomedicine. In addition, the functionalization of self-assembled peptide nanomaterials with other additive components enhances their stimuli-responsive functions, promoting function-specific applications that induced by both internal and external stimulations. In this review, we demonstrate recent advance in the peptide molecular design, self-assembly, functional tailoring, and biomedical applications of peptide-based nanomaterials. The strategies on the design and synthesis of single, dual, and multiple stimuli-responsive peptide-based nanomaterials with various dimensions are analyzed, and the functional regulation of peptide nanomaterials with active components such as metal/metal oxide, DNA/RNA, polysaccharides, photosensitizers, 2D materials, and others are discussed. In addition, the designed peptide-based nanomaterials with temperature-, pH-, ion-, light-, enzyme-, and ROS-responsive abilities for drug delivery, bioimaging, cancer therapy, gene therapy, antibacterial, as well as wound healing and dressing applications are presented and discussed. This comprehensive review provides detailed methodologies and advanced techniques on the synthesis of peptide nanomaterials from molecular biology, materials science, and nanotechnology, which will guide and inspire the molecular level design of peptides with specific and multiple functions for function-specific applications.

FKBP38 Regulates Self-Renewal and Survival of GBM Neurospheres.

Glioblastoma is the most common malignant primary brain tumor. The outcome is dismal, despite the multimodal therapeutic approach that includes surgical resection, followed by radiation and chemotherapy. The quest for novel therapeutic targets to treat glioblastoma is underway. FKBP38, a member of the immunophilin family of proteins, is a multidomain protein that plays an important role in the regulation of cellular functions, including apoptosis and autophagy. In this study, we tested the role of FKBP38 in glioblastoma tumor biology. Expression of FKBP38 was upregulated in the patient-derived primary glioblastoma neurospheres (GBMNS), compared to normal human astrocytes. Attenuation of FKBP38 expression decreased the viability of GBMNSs and increased the caspase 3/7 activity, indicating that FKBP38 is required for the survival of GBMNSs. Further, the depletion of FKBP38 significantly reduced the number of neurospheres that were formed, implying that FKBP38 regulates the self-renewal of GBMNSs. Additionally, the transient knockdown of FKBP38 increased the LC3-II/I ratio, suggesting the induction of autophagy with the depletion of FKBP38. Further investigation showed that the negative regulation of autophagy by FKBP38 in GBMNSs is mediated through the JNK/C-Jun-PTEN-AKT pathway. In vivo, FKBP38 depletion significantly extended the survival of tumor-bearing mice. Overall, our results suggest that targeting FKBP38 imparts an anti-glioblastoma effect by inducing apoptosis and autophagy and thus can be a potential therapeutic target for glioblastoma therapy.

Smart Orthopedic Biomaterials and Implants.

Musculoskeletal injuries including bone defects continue to present a significant challenge in orthopedic surgery due to suboptimal healing. Bone reconstruction strategies focused on the use of biological grafts and bone graft substitutes in the form of biomaterials-based 3D structures in fracture repair. Recent advances in biomaterials science and engineering have resulted in the creation of intricate 3D bone-mimicking structures that are mechanically stable, biodegradable, and bioactive to support bone regeneration. Current efforts are focused on improving the biomaterial and implant physicochemical properties to promote interactions with the host tissue and osteogenesis. The "smart" biomaterials and their 3D structures are designed to actively interact with stem/progenitor cells and the extracellular matrix (ECM) to influence the local environment towards osteogenesis and de novo tissue formation. This article will summarize such smart biomaterials and the methodologies to apply either internal or external stimuli to control the tissue healing microenvironment. A particular emphasis is also made on the use of smart biomaterials and strategies to create functional bioactive implants for bone defect repair and regeneration.

Targeting protein arginine methyltransferase 5 sensitizes glioblastoma to trametinib.

Background: The prognosis of glioblastoma (GBM) remains dismal because therapeutic approaches have limited effectiveness. A new targeted treatment using MEK inhibitors, including trametinib, has been proposed to improve GBM therapy. Trametinib had a promising preclinical effect against several cancers, but its adaptive treatment resistance precluded its clinical translation in GBM. Previously, we have demonstrated that protein arginine methyltransferase 5 (PRMT5) is upregulated in GBM and its inhibition promotes apoptosis and senescence in differentiated and stem-like tumor cells, respectively. We tested whether inhibition of PRMT5 can enhance the efficacy of trametinib against GBM. Methods: Patient-derived primary GBM neurospheres (GBMNS) with transient PRMT5 knockdown were treated with trametinib and cell viability, proliferation, cell cycle progression, ELISA, and western blot were analyzed. In vivo, NSG mice were intracranially implanted with PRMT5-intact and -depleted GBMNS, treated with trametinib by daily oral gavage, and observed for tumor progression and mice survival rate. Results: PRMT5 depletion enhanced trametinib-induced cytotoxicity in GBMNS. PRMT5 knockdown significantly decreased trametinib-induced AKT and ERBB3 escape pathways. However, ERBB3 inhibition alone failed to block trametinib-induced AKT activity suggesting that the enhanced antitumor effect imparted by PRMT5 knockdown in trametinib-treated GBMNS resulted from AKT inhibition and not ERBB3 inhibition. In orthotopic murine xenograft models, PRMT5-depletion extended the survival of tumor-bearing mice, and combination with trametinib further increased survival. Conclusion: Combined PRMT5/MEK inhibition synergistically inhibited GBM in animal models and is a promising strategy for GBM therapy.

Insulin-Functionalized Bioactive Fiber Matrices with Bone Marrow-Derived Stem Cells in Rat Achilles Tendon Regeneration.

Approximately half of annual musculoskeletal injuries in the US involve tendon tears. The naturally hypocellular and hypovascular tendon environment makes tendons injury-prone and heal slowly. Tendon tissue engineering strategies often use biomimetic scaffolds combined with bioactive factors and/or cells to enhance healing. FDA-approved growth factors to promote tendon healing are lacking, which highlights the need for safe and effective bioactive factors. Our previous work evaluated insulin as a bioactive factor and identified an optimal dose to promote in vitro mesenchymal stem cell survival, division, and tenogenesis. The present work evaluates the ability of insulin-functionalized electrospun nanofiber matrices with or without mesenchymal stem cells to enhance tendon repair in a rat Achilles injury model. Electrospun nanofiber matrices were functionalized with insulin, cultured with or without mesenchymal stem cells, and sutured to transected Achilles tendons in rats. We analyzed rat tendons 4 and 8 weeks after surgery for the tendon morphology, collagen production, and mechanical properties. Bioactive insulin-functionalized fiber matrices with mesenchymal stem cells resulted in significantly increased collagen I and III at 4 and 8 weeks postsurgery. Additionally, these matrices supported highly aligned collagen fibrils in the regenerated tendon tissue at 8 weeks. However, treatment- and control-regenerated tissues had similar tensile properties at 8 weeks, which were less than that of the native Achilles tendon. Our preliminary results establish the benefits of insulin-functionalized fiber matrices in promoting higher levels of collagen synthesis and alignment needed for functional recovery of tendon repair.

Subacromial bursa increases the failure force in a mouse model of supraspinatus detachment and repair.

BACKGROUND: It has been shown that subacromial bursa (SAB) harbors connective tissue progenitor cells. The purpose of this study was to evaluate the effects of implantation of SAB-derived cells (SBCs) suspended in a fibrin sealant bead and implantation of SAB tissue at rotator cuff repair site on biomechanical properties of the repair in a mouse (C57Bl/6) model of supraspinatus tendon (ST) detachment and repair. METHODS: Part 1: Murine SAB tissue was harvested and cultured. Viability of SBCs suspended in 10 µL of fibrin sealant beads was confirmed in vitro and in vivo. Eighty mice underwent right ST detachment and repair augmented with either fibrin sealant bead (control group) or fibrin sealant bead with 100,000 SBCs (study group) applied at the repair site. Part 2: 120 mice underwent right ST detachment and repair and were randomized equally into 4 groups: (1) a tissue group, which received a piece of freshly harvested SAB tissue; (2) a cell group, which received SBCs suspended in fibrin sealant bead; (3) a fibrin sealant group, which received plain fibrin sealant bead without cells; and (4) a control group, which received nothing at the ST repair site. An equal number of mice in each group were killed at 2 and 4 weeks. Specimens underwent biomechanical testing to evaluate failure force (part 1 and 2) and histologic analysis of the repair site (part 1 only). RESULTS: Part 1: The mean failure force in the study group was significantly higher than controls at 2 and 4 weeks (3.25 ± 1.03 N vs. 2.43 ± 0.56 N, P = .01, and 4.08 ± 0.99 N vs. 3.02 ± 0.8 N, P = .004, respectively). Mean cell density of the ST at the repair site was significantly lower in the study group at 2 weeks than in controls (18,292.13 ± 1706.41 vs. 29,501.90 ± 3627.49, P = .001). Study group specimens had lower proteoglycan contents than controls, but this difference was not statistically significant. Part 2: There was no difference in failure force between cell and tissue groups at the 2- and 4-week time points (P = .994 and P = .603, respectively). There was no difference in failure force between fibrin sealant bead and control groups at the 2- and 4-week time points (P = .978 and P = .752, respectively). CONCLUSION: This study shows that the application of SBCs and SAB tissue at the rotator cuff repair site increases the strength of repair in a murine model of rotator cuff detachment and repair.

Characterization of murine subacromial bursal-derived cells.

PURPOSE/AIM: The purpose of this study is to identify a cell population within the murine subcromial bursal-derived cells with characteristics compatible to an accepted mesenchymal stem cell description given by the International Society for Cellular Therapy (ISCT). MATERIALS AND METHODS: Murine subacromial bursa was harvested using microsurgical technique. Subacromial bursal-derived cells were classified through colony-forming units, microscopic morphology, fluorescent-activated cell sorting, and differentiation into chondrogenic, adipogenic, and osteogenic lineages. RESULTS: Subacromial bursal samples exhibited cell growth out of the tissue for an average of 115 ± 29 colony-forming units per 1 mL of complete media. Subacromial bursal-derived cells exhibited a long, spindle-shaped, fibroblast-like morphology. Subacromial bursal-derived cells positively expressed mesenchymal stem cell markers CD73, CD90, and CD105, and negatively expressed mesenchymal stem cell markers CD31 and CD45. Subacromial bursal-derived cells, examined by Image J analysis and quantitative gene expression, were found to differentiate into chondrogenic, adipogenic, and osteogenic lineages. CONCLUSIONS: This study demonstrated the feasibility of harvesting murine subacromial bursal tissue and identified a cell population within the subacromial bursa with characteristics compatible to an accepted mesenchymal stem cell description. The results of this study suggest that the mouse subacromial bursal-derived cell population harbors mesenchymal stem cells. Murine subacromial bursal tissue is a potential source for obtaining cells with mesenchymal stem cell characteristics for future utilization in orthopedic research to look into treatment of rotator cuff pathology.

The glucagon-like peptide 1 receptor agonist Exendin-4 induces tenogenesis in human mesenchymal stem cells.

Tendon injuries are common and account for up to 50% of musculoskeletal injuries in the United States. The poor healing nature of the tendon is attributed to poor vascularization and cellular composition. In the absence of FDA-approved growth factors for tendon repair, engineering strategies using bioactive factors, donor cells, and delivery matrices to promote tendon repair and regeneration are being explored. Growth factor alternatives in the form of small molecules, donor cells, and progenitors offer several advantages and enhance the tendon healing response. Small drug molecules and peptides offer stability over growth factors that are known to suffer from relatively short biological half-lives. The primary focus of this study was to assess the ability of the exendin-4 (Ex-4) peptide, a glucagon-like peptide 1 (GLP-1) receptor agonist, to induce tenocyte differentiation in bone marrow-derived human mesenchymal stem cells (hMSCs). We treated hMSCs with varied doses of Ex-4 in culture media to evaluate proliferation and tendonogenic differentiation. A 20 nM Ex-4 concentration was optimal for promoting cell proliferation and tendonogenic differentiation. Tendonogenic differentiation of hMSCs was evaluated via gene expression profile, immunofluorescence, and biochemical analyses. Collectively, the levels of tendon-related transcription factors (Mkx and Scx) and extracellular matrix (Col-I, Dcn, Bgn, and Tnc) genes and proteins were elevated compared to media without Ex-4 and other controls including insulin and IGF-1 treatments. The tendonogenic factor Ex-4 in conjunction with hMSCs appear to enhance tendon regeneration.

Evaluation of Autologously Derived Biomaterials and Stem Cells for Bone Tissue Engineering.

Despite progress, clinical translation of tissue engineering (TE) products/technologies is limited. A significant effort is underway to develop biomaterials and cells through a minimally modified process for clinical translation of TE products. Recently, bone marrow aspirate (BMA) was identified as an autologous source of cells for TE applications and is currently being tested in clinical therapies, but the isolation methods need improvement to avoid potential for contamination and increase progenitor cell yield. To address these issues, we reproducibly processed human peripheral blood (PB) and BMA to develop autologously derived biomaterials and cells. We demonstrated PB-derived biomaterial/gel cross-linking and fibrin gel formation with varied gelation times as well as biocompatibility through support of human bone marrow-derived stem cell survival and growth in vitro. Next, we established a plastic culture-free process that concentrates and increases the yield of CD146+/CD271+ early mesenchymal progenitor cells in BMA (concentrated BMA [cBMA]). cBMA exhibited increased colony formation and multipotency (including chondrogenic differentiation) in vitro compared with standard BMA. PB-derived gels encapsulated with cBMA also demonstrated increased cell proliferation and enhanced mineralization when assessed for bone TE in vitro. This strategy can potentially be developed for use in any tissue regeneration application; however, bone regeneration was used as a test bed for this study.

Polymeric nanofibrous nerve conduits coupled with laminin for peripheral nerve regeneration.

Artificial nerve guidance conduits (NGCs) are being investigated as an alternative to autografts, since autografts are limited in supply. A polycaprolactone (PCL)-based spiral NGC with crosslinked laminin on aligned nanofibers was evaluated in vivo post a successful in vitro assessment. PC-12 cell assays confirmed that the aligned nanofibers functionalized with laminin were able to guide and enhance neurite outgrowth. In the rodent model, the data demonstrated that axons were able to regenerate across the critical nerve gap, when laminin was present. Without laminin, the spiral NGC with aligned nanofibers group resulted in a random cluster of extracellular matrix tissue following injuries. The reversed autograft group performed best, showing the most substantial improvement based on nerve histological assessment and gastrocnemius muscle measurement. Nevertheless, the recovery time was too short to obtain meaningful data for the motor functional assessments. A full motor recovery may take up to years. An interesting observation was noted in the crosslinked laminin group. Numerous new blood capillary-like structures were found around the regenerated nerve. Owing to recent studies, we hypothesized that new blood vessel formation could be one of the key factors to increase the successful rate of nerve regeneration in the current study. Overall, these findings indicated that the incorporation of laminin into polymeric nerve conduits is a promising strategy for enhancing peripheral nerve regeneration. However, the best combination of contact-guidance cues, haptotactic cues, and chemotactic cues have yet to be realized. The natural sequence of nerve regeneration should be studied more in-depth before modulating any strategies.

Assessment of Hedgehog Signaling Pathway Activation for Craniofacial Bone Regeneration in a Critical-Sized Rat Mandibular Defect.

IMPORTANCE: Osseous craniofacial defects are currently reconstructed with bone grafting, rigid fixation, free tissue transfer, and/or recombinant human bone morphogenetic protein 2. Although these treatment options often have good outcomes, they are associated with substantial morbidity, and many patients are not candidates for free tissue transfer. OBJECTIVE: To assess whether polysaccharide-based scaffold (PS) constructs that are cross-linked with smoothened agonist (SAG), vascular endothelial growth factor (VEGF), and bone morphogenetic protein 6 (BMP-6) would substantially increase bone regeneration. DESIGN, SETTING, AND PARTICIPANTS: This animal model study was conducted at the University of Virginia School of Medicine Cui Laboratory from March 1, 2017, to June 30, 2017. Thirty-three 10-week-old female Lewis rats were acquired for the study. Bilateral nonsegmental critical-sized defects were created in the angle of rat mandibles. The defects were either left untreated or filled with 1 of the 9 PSs. The rats were killed after 8 weeks, and bone regeneration was evaluated using microcomputed tomographic imaging and mechanical testing. Analysis of variance testing was used to compare the treatment groups. MAIN OUTCOMES AND MEASURES: Blinded analysis and computer analysis of the microcomputed tomographic images were used to assess bone regeneration. RESULTS: In the 33 female Lewis rats, minimal healing was observed in the untreated mandibles. Addition of SAG was associated with increases in bone regeneration and bone density in all treatment groups, and maximum bone healing was seen in the group with BMP-6, VEGF, and SAG cross-linked to PS. For each of the 5 no scaffold group vs BMP-6, VEGF, and SAG cross-linked to PS group comparisons, mean defect bone regeneration was 4.14% (95% CI, 0.94%-7.33%) vs 66.19% (95% CI, 54.47%-77.90%); mean bone volume, 14.52 mm3 (95% CI, 13.07-15.97 mm3) vs 20.87 mm3 (95% CI, 14.73- 27.01 mm3); mean bone surface, 68.97 mm2 (95% CI, 60.08-77.85 mm2) vs 96.77 mm2 (95% CI, 76.11-117.43 mm2); mean ratio of bone volume to total volume, 0.11 (95% CI, 0.10-0.11) vs 0.15 (95% CI, 0.10-0.19); and mean connectivity density 0.03 (95% CI, 0.02-0.05) vs 0.32 (95% CI, 0.25-0.38). On mechanical testing, mandibles with untreated defects broke with less force than control mandibles in which no defect was made, although this force did not reach statistical significance. No significant difference in force to fracture was observed among the treatment groups. CONCLUSIONS AND RELEVANCE: In this rat model study, activation of the hedgehog signaling pathway using smoothened agonist was associated with increased craniofacial bone regeneration compared with growth factors alone, including US Food and Drug Administration-approved recombinant human bone morphogenetic protein 2. Pharmaceuticals that target this pathway may offer a new reconstructive option for bony craniofacial defects as well as nonunion and delayed healing fractures. LEVEL OF EVIDENCE: NA.

Polymeric ionically conductive composite matrices and electrical stimulation strategies for nerve regeneration: In vitro characterization.

Stem cell strategies and the use of electrical stimulation (ES) represent promising new frontiers for peripheral nerve regeneration. Composite matrices were fabricated by coating electrospun polycaprolactone/cellulose acetate micro-nanofibers with chitosan and ionically conductive (IC) polymers including, sulfonated polyaniline, and lignin sulfonate. These composite matrices were characterized for surface morphology, coating uniformity, ionic conductivity, and mechanical strength to explore as scaffold materials for nerve regeneration in conjunction with ES. Composite matrices measured conductivity in the range of 0.0049-0.0068 mS/m due to the uniform coating of sulfonated polymers on the micro-nanofibers. Thin films (2D) and composite fiber matrices (3D) of IC polymers seeded with human mesenchymal stem cells (hMSCs) were electrically stimulated at 0.5 V, 20 Hz for 1 h daily for 14 days to study the changes in cell viability, morphology, and expression of the neuronal-like phenotype. In vitro ES lead to changes in hMSCs' fibroblast morphology into elongated neurite-like structures with cell bodies for ES-treated and positive control growth factor-treated groups. Immunofluorescent staining revealed the presence of neuronal markers including ß3-tubulin, microtubule-associated protein 2, and nestin in response to ES. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1792-1805, 2019.

Nanomaterials/Nanocomposites for Osteochondral Tissue.

For many years, the avascular nature of cartilage tissue has posed a clinical challenge for replacement, repair, and reconstruction of damaged cartilage within the human body. Injuries to cartilage and osteochondral tissues can be due to osteoarthritis, sports, aggressive cancers, and repetitive stresses and inflammation on wearing tissue. Due to its limited capacity for regeneration or repair, there is a need for suitable material systems which can recapitulate the function of the native osteochondral tissue physically, mechanically, histologically, and biologically. Tissue engineering (TE) approaches take advantage of principles of biomedical engineering, clinical medicine, and cell biology to formulate, functionalize, and apply biomaterial scaffolds to aid in the regeneration and repair of tissues. Nanomaterial science has introduced new methods for improving and fortifying TE scaffolds, and lies on the forefront of cutting-edge TE strategies. These nanomaterials enable unique properties directly correlated to their sub-micron dimensionality including structural and cellular advantages. Examples include electrospun nanofibers and emulsion nanoparticles which provide nanoscale features for biomaterials, more closely replicating the 3D extracellular matrix, providing better cell adhesion, integration, interaction, and signaling. This chapter aims to provide a detailed overview of osteochondral regeneration and repair using TE strategies with a focus on nanomaterials and nanocomposites.

Micro-Nanostructures of Cellulose-Collagen for Critical Sized Bone Defect Healing.

Bone tissue engineering strategies utilize biodegradable polymeric matrices alone or in combination with cells and factors to provide mechanical support to bone, while promoting cell proliferation, differentiation, and tissue ingrowth. The performance of mechanically competent, micro-nanostructured polymeric matrices, in combination with bone marrow stromal cells (BMSCs), is evaluated in a critical sized bone defect. Cellulose acetate (CA) is used to fabricate a porous microstructured matrix. Type I collagen is then allowed to self-assemble on these microstructures to create a natural polymer-based, micro-nanostructured matrix (CAc). Poly (lactic-co-glycolic acid) matrices with identical microstructures serve as controls. Significantly higher number of implanted host cells are distributed in the natural polymer based micro-nanostructures with greater bone density and more uniform cell distribution. Additionally, a twofold increase in collagen content is observed with natural polymer based scaffolds. This study establishes the benefits of natural polymer derived micro-nanostructures in combination with donor derived BMSCs to repair and regenerate critical sized bone defects. Natural polymer based materials with mechanically competent micro-nanostructures may serve as an alternative material platform for bone regeneration.

Engineered stem cell niche matrices for rotator cuff tendon regenerative engineering.

Rotator cuff (RC) tears represent a large proportion of musculoskeletal injuries attended to at the clinic and thereby make RC repair surgeries one of the most widely performed musculoskeletal procedures. Despite the high incidence rate of RC tears, operative treatments have provided minimal functional gains and suffer from high re-tear rates. The hypocellular nature of tendon tissue poses a limited capacity for regeneration. In recent years, great strides have been made in the area of tendonogenesis and differentiation towards tendon cells due to a greater understanding of the tendon stem cell niche, development of advanced materials, improved scaffold fabrication techniques, and delineation of the phenotype development process. Though in vitro models for tendonogenesis have shown promising results, in vivo models have been less successful. The present work investigates structured matrices mimicking the tendon microenvironment as cell delivery vehicles in a rat RC tear model. RC injuries augmented with a matrix delivering rat mesenchymal stem cells (rMSCs) showed enhanced regeneration over suture repair alone or repair with augmentation, at 6 and 12-weeks post-surgery. The local delivery of rMSCs led to increased mechanical properties and improved tissue morphology. We hypothesize that the mesenchymal stem cells function to modulate the local immune and bioactivity environment through autocrine/paracrine and/or cell homing mechanisms. This study provides evidence for improved tendon healing with biomimetic matrices and delivered MSCs with the potential for translation to larger, clinical animal models. The enhanced regenerative healing response with stem cell delivering biomimetic matrices may represent a new treatment paradigm for massive RC tendon tears.

Electrospun Nanofiber Scaffolds and Their Hydrogel Composites for the Engineering and Regeneration of Soft Tissues.

Electrospinning has emerged as a simple, elegant, and scalable technique that can be used to fabricate polymeric nanofibers. Pure polymers as well as blends and composites of both natural and synthetic ones have been successfully electrospun into nanofiber matrices for many biomedical applications. Tissue-engineered medical implants, such as polymeric nanofiber scaffolds, are potential alternatives to autografts and allografts, which are short in supply and carry risks of disease transmission. These scaffolds have been used to engineer various soft tissues, including connective tissues, such as skin, ligament, and tendon, as well as nonconnective ones, such as vascular, muscle, and neural tissue. Electrospun nanofiber matrices show morphological similarities to the natural extracellular matrix (ECM), characterized by ultrafine continuous fibers, high surface-to-volume ratios, high porosities, and variable pore-size distributions. The physiochemical properties of nanofiber matrices can be controlled by manipulating electrospinning parameters so that they meet the requirements of a specific application.Nanostructured implants show improved biological performance over bulk materials in aspects of cellular infiltration and in vivo integration, taking advantage of unique quantum, physical, and atomic properties. Furthermore, the topographies of such scaffolds has been shown to dictate cellular attachment, migration, proliferation, and differentiation, which are critical in engineering complex functional tissues with improved biocompatibility and functional performance. This chapter discusses the use of the electrospinning technique in the fabrication of polymer nanofiber scaffolds utilized for the regeneration of soft tissues. Selected scaffolds will be seeded with human mesenchymal stem cells (hMSCs), imaged using scanning electron and confocal microscopy, and then evaluated for their mechanical properties as well as their abilities to promote cell adhesion, proliferation , migration, and differentiation.

Guided differentiation of bone marrow stromal cells on co-cultured cartilage and bone scaffolds.

Focal chondral defects that result from traumatic injuries to the knee remain one of the most common causes of disability in patients. Current solutions for healing focal cartilage defects are mainly limited by the production of inferior cartilage-like tissue and subsequent delamination due to incomplete healing of the subchondral bone. In this experiment a polymeric osteochondral implant for guiding autologous bone marrow stem cells (BMSCs) to populate the scaffold to create distinctive bone and cartilage tissue is used. The cartilage component presents bioactive aligned nanofibers containing chondroitin sulfate and hyaluronic acid while the bone component includes hydroxyapatite to promote chondrogenic and osteogenic differentiation of the rat BMSCs in vitro. The different cartilage and bone components resulted in the elevated expression of osteogenic markers such as bone sialoprotein, runt related transcription factor 2, and bone morphogenetic protein 2 in the deeper bone layer and chondrogenic markers such as collagen type II and aggrecan in the cartilage layer. Through immunofluorescence imaging, the alignment of the secreted collagen type II fibrils and aggrecan was visualized and quantified on the cartilage component of the scaffold. These current studies show that the biodegradable biphasic osteochondral implant may be effective in promoting more hyaline-like tissue to fill in chondral defects of the knee.

Influence of chondroitin sulfate and hyaluronic acid presence in nanofibers and its alignment on the bone marrow stromal cells: cartilage regeneration.

Cartilage degeneration is the major cause of disability and poses several challenges to repair and regenerate. Conventional surgical treatments often induce fibrous tissues and compromise its function. Alternative tissue engineering strategies utilized scaffolds, factors and cells alone or in combination with some degree of success. This study reports the use of nanostructured biomimetic scaffold system in regulating the rat bone marrow stem cells (rBMSCs) differentiation into chondrogenic lineage in vitro. The biometric scaffold is essentially a micro-porous polycaprolactone (PCL) spiral structure decorated with sparsely spaced bioactive PCL nanofibers. The bioactivity stems from the use of two major components of hyaline cartilage extracellular matrix (ECM) namely chondroitin sulfate (CS) and hyaluronic acid (HYA). The PCL spiral structure was surface functionalized with PCL nanofibers encapsulated with CS (20% (w/w)) and HYA (0.2% (w/w)). In order to retain and sustain the release of CS and HYA nanofibers were cross-linked using carbodiimide chemistry. This study also evaluated the effect of nanofiber alignment on rBMSCs differentiation and evaluated the production of characteristic hyaline cartilage proteins namely collagen type II and aggrecan in vitro up to 28 days. Rat bone marrow derived stem cells cultured on the aligned nanofibers expressed significantly elevated levels of collagen type II and aggrecan secretions (western blots) as compared to scaffolds decorated with random fibers and tissue culture polystyrene (TCPS). This fiber alignment dependent expression of collagen type II and aggrecan secretion were further confirmed through immunofluorescence staining. This biomimetic and bioactive scaffold may serve as a serve as an efficient scaffold system for cartilage regeneration.

Collagen functionalized bioactive nanofiber matrices for osteogenic differentiation of mesenchymal stem cells: bone tissue engineering.

Scaffold architecture, surface topography, biochemical and mechanical cues have been shown to significantly improve cellular events and in vivo tissue regeneration. Specifically electrospun nanofiber matrices have gained tremendous interest due to their intrinsic structural resemblance to native tissue extracellular matrix (ECM). The present study reports on the electrospun nanofiber matrices of polycaprolactone (PCL)-chitosan (CS) blends and effect of type I collagen surface functionalization in regulating rat bone marrow derived stromal cells (rBMSCs) differentiation into osteogenic lineage. Collagen was covalently attached to blend nanofibers via carbodiimide (EDC) coupling. Bead-free smooth nanofibers (diameter-700-850 nm) obtained at the optimized conditions of polymer concentration and electrospinning parameters were used for the study. EDC collagen coupling resulted in 0.120+/-0.016 micro g of collagen immobilization onto a 1 cm2 area of the PCL/CS nanofibers, which was 2.6-folds higher than the amount of collagen that can be retained by physical adsorption. Significantly improved rBMSCs adhesion, spreading, proliferation and osteogenic differentiation was observed on the collagen functionalized COL-PCULCS nanofiber matrices as compared to control groups. Osteogenic phenotypic markers such as alkaline phosphatase (ALP) activity and mineralization were found to be significantly higher on COL-PCL/CS nanofiber matrices as compared to controls. Elevated gene expression profiles of osteogenic markers such as osteocalcin (0CN), osteopontin (OPN) and ALP further corroborate the osteoinductive nature of the collagen functionalized PCL/CS nanofiber matrices. These fiber matrices and modification techniques could be extended to other scaffold systems for tissue engineering applications.

Properties of biologic scaffolds and their response to mesenchymal stem cells.

PURPOSE: The purpose of this study was to examine, in vitro, the cellular response of human mesenchymal stem cells (MSCs) to sample types of commercially available scaffolds in comparison with control, native tendon tissue (fresh-frozen rotator cuff tendon allograft). METHODS: MSCs were defined by (1) colony-forming potential; (2) ability to differentiate into tendon, cartilage, bone, and fat tissue; and (3) fluorescence-activated cell sorting analysis (CD73, CD90, CD45). Samples were taken from fresh-frozen human rotator cuff tendon (allograft), human highly cross-linked collagen membrane (Arthroflex; LifeNet Health, Virginia Beach, VA), porcine non-cross-linked collagen membrane (Mucograft; Geistlich Pharma, Lucerne, Switzerland), a human platelet-rich fibrin matrix (PRF-M), and a fibrin matrix based on platelet-rich plasma (ViscoGel; Arthrex, Naples, FL). Cells were counted for adhesion (24 hours), thymidine assay for cell proliferation (96 hours), and live/dead stain for viability (168 hours). Histologic analysis was performed after 21 days, and the unloaded scaffolds were scanned with electron microscopy. RESULTS: MSCs were successfully differentiated into all cell lines. A significantly greater number of cells adhered to both the non-cross-linked porcine collagen scaffold and PRF-M. Cell activity (proliferation) was significantly higher in the non-cross-linked porcine collagen scaffold compared with PRF-M and fibrin matrix based on platelet-rich plasma. There were no significant differences found in the results of the live/dead assay. CONCLUSIONS: Significant differences in the response of human MSCs to biologic scaffolds existed. MSC adhesion, proliferation, and scaffold morphology evaluated by histologic analysis and electron microscopy varied throughout the evaluated types of scaffolds. Non-cross-linked porcine collagen scaffolds showed superior results for cell adhesion and proliferation, as well as on histologic evaluation. CLINICAL RELEVANCE: This study enables the clinician and scientist to choose scaffold materials according to their specific interaction with MSCs.