Scaffold For Tendon Tissue Engineering Research Articles

Stem Cell‐Based Therapies and Tissue Engineering Innovations for Tendinopathy: A Comprehensive Review of Current Strategies and Future Directions

AbstractTendon diseases commonly lead to physical disability, exerting a profound impact on the routine of affected patients. These conditions respond poorly to existing treatments, presenting a substantial challenge for orthopedic scientists. Research into clinical translational therapy has yet to yield highly versatile interventions capable of effectively addressing tendon diseases, including tendinopathy. Stem cell‐based therapies have emerged as a promising avenue for modifying the biological milieu through the secretion of regenerative and immunomodulatory factors. The current review provides an overview of the intricate tendon microenvironment, encompassing various tendon stem progenitor cells within distinct tendon sublocations, gene regulation, and pathways pertinent to tendon development, and the pathology of tendon diseases. Subsequently, the advantages of stem cell‐based therapies are discussed that utilize distinct types of autologous and allogeneic stem cells for tendon regeneration at the translational level. Moreover, this review outlines the challenges, gaps, and future innovations to propose a consolidated stem cell‐based therapy to treat tendinopathy. Finally, regenerative soluble therapies, insoluble bio‐active therapies, along with insoluble bio‐active therapies, and implantable 3D scaffolds for tendon tissue engineering are discussed, thereby presenting a pathway toward enhanced tissue regeneration and engineering.

Braided suture-reinforced fibrous yarn bundles as a scaffold for tendon tissue engineering in extensor digitorum tendon repair

In this study, we intend to develop an effective tendon tissue engineering scaffold that can provide mechanical stability and tendon regeneration ability. Using a novel electrospinning process, a biodegradable suture was continuously covered with aligned polycaprolactone fibers in core/sheath structure to produce a single yarn. The single yarn was braided together to fabricate a multi yarn (MY) scaffold, which can be surface modified with oxygen plasma and conjugated with heparin. The fibroblast growth factor 2 (FGF2) was bound to MY through bioaffinity between heparin and FGF2 to generate a functional scaffold (MY-FGF2) suitable for extensor digitorum tendon (EDT) repair. The physico-chemical properties of the scaffolds were characterized throughout the modification steps using microscopy, spectroscopy and mechanical testing. In vitro static culture using rabbit tendon-derived fibroblasts (rTFs) indicates combined effects of FGF2 and fiber alignment can enhance cell proliferation and extracellular matrix synthesis rates, as well as fasten tendon maturation. The cytoskeleton staining further endorses aligned morphology of fibers direct cell growth and collagen fiber deposition along the fiber axial direction, mimicking native tendon features. The dynamic culture in a bioreactor under uniaxial cyclic tensile loading authenticates 5 % mechanical stimulation can further increase cell proliferation and tenogenic differentiation rates compared to static culture. After mechanical stimulation for 7 days in vitro, the MY-FGF2/rTFs construct was used for repair of EDT defects in rabbits. The retrieved MY-FGF2/rTFs sample 6-week post-implantation shows superior mechanical properties and tendon regeneration abilities over acellular MY-FGF2. Taken together, we demonstrate a combinatory approach with MY-FGF2 where chemical and physical cues provided by FGF2, fiber alignment and dynamic stimulation contribute to tendon regeneration with a specific focus on EDT repair.

Development of high resilience spiral wound suture-embedded gelatin/PCL/heparin nanofiber membrane scaffolds for tendon tissue engineering

This study develops a spiral wound scaffold based on gelatin/PCL/heparin (GPH) nanofiber membranes for tendon tissue engineering. By embedding sutures in dual layers of aligned GPH nanofiber membranes, prepared from mixed electrospinning of gelatin and PCL/heparin solutions, we fabricate a high resilience scaffold intended for the high loading environment experienced by tendons. The basic fibroblast growth factor (bFGF) was anchored to GPH scaffold through bioaffinity between heparin and bFGF, aim to provide biological cues for maintenance of tenogenic phenotype. In addition, the aligned nanofiber morphology is expected to provide physical cues toward seeded tenocytes. With sustained release of bFGF, GPH-bFGF can enhance proliferation, up-regulate tenogenic gene expression, and increase synthesis of tendon-specific proteins by tenocytes in vitro. Furthermore, by properly maintaining tendon phenotypes, GPH-bFGF/tenocytes constructs showed improved mechanical properties over GPH-bFGF. From in vivo study using GPH-bFGF/tenocytes constructs to repair rabbit Achilles tendon defects, neotendon tissue formation was confirmed from histological staining and biomechanical analysis. These findings collectively demonstrate that the newly designed GPH-bFGF scaffold could provide a niche for inducing tendon tissue regeneration by effectively restoring the tendon tissue structure and function.

The application of human periodontal ligament stem cells and biomimetic silk scaffold for in situ tendon regeneration

BackgroundWith the development of tissue engineering, enhanced tendon regeneration could be achieved by exploiting suitable cell types and biomaterials. The accessibility, robust cell amplification ability, superior tendon differentiation potential, and immunomodulatory effects of human periodontal ligament stem cells (hPDLSCs) indicate their potential as ideal seed cells for tendon tissue engineering. Nevertheless, there are currently no reports of using PDLSCs as seed cells. Previous studies have confirmed the potential of silk scaffold for tendon tissue engineering. However, the biomimetic silk scaffold with tendon extracellular matrix (ECM)-like structure has not been systematically studied for in situ tendon regeneration. Therefore, this study aims to evaluate the effects of hPDLSCs and biomimetic silk scaffold on in situ tendon regeneration.MethodsHuman PDLSCs were isolated from extracted wisdom teeth. The differentiation potential of hPDLSCs towards osteo-, chondro-, and adipo-lineage was examined by cultured in different inducing media. Aligned and random silk scaffolds were fabricated by the controlled directional freezing technique. Scaffolds were characterized including surface structure, water contact angle, swelling ratio, degradation speed and mechanical properties. The biocompatibility of silk scaffolds was evaluated by live/dead staining, SEM observation, cell proliferation determination and immunofluorescent staining of deposited collagen type I. Subsequently, hPDLSCs were seeded on the aligned silk scaffold and transplanted into the ruptured rat Achilles tendon. Scaffolds without cells served as control groups. After 4 weeks, histology evaluation was carried out and macrophage polarization was examined to check the repair effects and immunomodulatory effects.ResultsHuman PDLSCs were successfully isolated, and their multi-differentiation potential was confirmed. Compared with random scaffold, aligned silk scaffold had more elongated and aligned pores and promoted the proliferation and ordered arrangement of hPDLSCs. After implantation into rat Achilles tendon defect, hPDLSCs seeded aligned silk scaffold enhanced tendon repair with more tendon-like tissue formation after 4 weeks, as compared to the scaffold-only groups. Higher expression of CD206 and lower expression of iNOS, IL-1β and TNF-α were found in the hPDLSCs seeded aligned silk scaffold group, which revealed its modulation effect of macrophage polarization from M1 to M2 phenotype.ConclusionsIn summary, this study demonstrates the efficacy of hPDLSCs as seed cells and aligned silk scaffold as a tendon-mimetic scaffold for enhanced tendon tissue engineering, which may have broad implications for future tendon tissue engineering and regenerative medicine researches.

Tension Stimulation of Tenocytes in Aligned Hyaluronic Acid/Platelet-Rich Plasma-Polycaprolactone Core-Sheath Nanofiber Membrane Scaffold for Tendon Tissue Engineering.

To recreate the in vivo niche for tendon tissue engineering in vitro, the characteristics of tendon tissue underlines the use of biochemical and biophysical cues during tenocyte culture. Herein, we prepare core-sheath nanofibers with polycaprolactone (PCL) sheath for mechanical support and hyaluronic acid (HA)/platelet-rich plasma (PRP) core for growth factor delivery. Three types of core-sheath nanofiber membrane scaffolds (CSNMS), consisting of random HA-PCL nanofibers (Random), random HA/PRP-PCL nanofibers (Random+) or aligned HA/PRP-PCL (Align+) nanofibers, were used to study response of rabbit tenocytes to biochemical (PRP) and biophysical (fiber alignment) stimulation. The core-sheath structures as well as other pertinent properties of CSNMS have been characterized, with Align+ showing the best mechanical properties. The unidirectional growth of tenocytes, as induced by aligned fiber topography, was confirmed from cell morphology and cytoskeleton expression. The combined effects of PRP and fiber alignment in Align+ CSNMS lead to enhanced cell proliferation rates, as well as upregulated gene expression and marker protein synthesis. Another biophysical cue on tenocytes was introduced by dynamic culture of tenocyte-seeded Align+ in a bioreactor with cyclic tension stimulation. Augmented by this biophysical beacon from mechanical loading, dynamic cell culture could shorten the time for tendon maturation in vitro, with improved cell proliferation rates and tenogenic phenotype maintenance, compared to static culture. Therefore, we successfully demonstrate how combined use of biochemical/topographical cues as well as mechanical stimulation could ameliorate cellular response of tenocytes in CSNMS, which can provide a functional in vitro environmental niche for tendon tissue engineering.

The Role of Scaffolds in Tendon Tissue Engineering.

Tendons are unique forms of connective tissue aiming to transmit the mechanical force of muscle contraction to the bones. Tendon injury may be due to direct trauma or might be secondary to overuse injury and age-related degeneration, leading to inflammation, weakening and subsequent rupture. Current traditional treatment strategies focus on pain relief, reduction of the inflammation and functional restoration. Tendon repair surgery can be performed in people with tendon injuries to restore the tendon’s function, with re-rupture being the main potential complication. Novel therapeutic approaches that address the underlying pathology of the disease is warranted. Scaffolds represent a promising solution to the challenges associated with tendon tissue engineering. The ideal scaffold for tendon tissue engineering needs to exhibit physiologically relevant mechanical properties and to facilitate functional graft integration by promoting the regeneration of the native tissue.

Mechanical and Biochemical Stimulation of 3D Multilayered Scaffolds for Tendon Tissue Engineering.

Tendon injuries are frequent and occur in the elderly, young, and athletic populations. The inadequate number of donors combined with many challenges associated with autografts, allografts, xenografts, and prosthetic devices have added to the value of engineering biological substitutes, which can be implanted to repair the damaged tendons. Electrospun scaffolds have the potential to mimic the native tissue structure along with desired mechanical properties and, thus, have attracted noticeable attention. In order to improve the biological responses of these fibrous structures, we designed and fabricated 3D multilayered composite scaffolds, where an electrospun nanofibrous substrate was coated with a thin layer of cell-laden hydrogel. The whole construct composition was optimized to achieve adequate mechanical and physical properties as well as cell viability and proliferation. Mesenchymal stem cells (MSCs) were differentiated by the addition of bone morphogenetic protein 12 (BMP-12). To mimic the natural function of tendons, the cell-laden scaffolds were mechanically stimulated using a custom-built bioreactor. The synergistic effect of mechanical and biochemical stimulation was observed in terms of enhanced cell viability, proliferation, alignment, and tenogenic differentiation. The results suggested that the proposed constructs can be used for engineering functional tendons.

Insulin immobilized PCL-cellulose acetate micro-nanostructured fibrous scaffolds for tendon tissue engineering.

Use of growth factors as biochemical molecules to elicit cellular differentiation is a common strategy in tissue engineering. However, limitations associated with growth factors, such as short half-life, high effective physiological doses, and high costs, have prompted the search for growth factor alternatives, such as growth factor mimics and other proteins. This work explores the use of insulin protein as a biochemical factor to aid in tendon healing and differentiation of cells on a biomimetic electrospun micro-nanostructured scaffold. Dose response studies were conducted using human mesenchymal stem cells (MSCs) in basal media supplemented with varied insulin concentrations. A dose of 100-ng/mL insulin showed increased expression of tendon markers. Synthetic-natural blends of various ratios of polycaprolactone (PCL) and cellulose acetate (CA) were used to fabricate micro-nanofibers to balance physicochemical properties of the scaffolds in terms of mechanical strength, hydrophilicity, and insulin delivery. A 75:25 ratio of PCL:CA was found to be optimal in promoting cellular attachment and insulin immobilization. Insulin insulin deliveryimmobilized fiber matrices also showed increased expression of tendon phenotypic markers by MSCs similar to findings with insulin supplemented media, indicating preservation of insulin bioactivity. Insulin functionalized scaffolds may have potential applications in tendon healing and regeneration.

Anisotropic cytocompatible electrospun scaffold for tendon tissue engineering elicits limited inflammatory response in vitro

Tendon tears are a relevant concern for today's national health systems because of their social impact and high recurrence rate. The current gold standard for fixing tendon tears is surgical repair; however, this strategy is not able to fully re-establish tendon integrity and functionality. Tissue engineering approaches aim at promoting tissue regeneration by delivering the opportune signals to the injured site combining biomaterials, cells and biochemical cues. Electrospinning is currently one of the most versatile polymer processing techniques that allows manufacturing of nano- and micro-fibres substrates. Such fibrous morphology is deemed to be an ideal substrate to convey topographical cues to cells. Here we evaluated the potential of polycaprolactone processed by means of electrospinning technology for tendon tissue engineering. Fibrous free-of-defects substrate with random and aligned fibres were successfully fabricated. Rat tenocytes were used to assess the cytocompatibility of the substrates for application as tendon tissue engineered devices. Tenocytes were able to proliferate and adapt to the substrates topography acquiring an elongated morphology, which is the precondition for oriented collagen deposition, when seeded on aligned fibres. Real time Polymerase Chain Reaction (Rt-PCR) also revealed the overall maintenance of tenocyte phenotype over 7 days culture. To verify suitability for in vivo implantation, the level of inflammatory cytokine genes expressed by THP-1 cells cultured in presence of electrospun polycaprolactone substrates was evaluated. Inflammatory response was limited. The novel preliminary in vitro work presented herein showing tenocytes compatibility and limited inflammatory cytokines synthesis suggests that electrospun polycaprolactone may be taken into consideration as substrate for tendon healing applications.

An asymmetric chitosan scaffold for tendon tissue engineering: In vitro and in vivo evaluation with rat tendon stem/progenitor cells

The poor healing capacity and typically incomplete regeneration of injured tendons has made tendon repair as a primary clinical concern. Several methods for repairing injured tendons have been developed in the last decade. Tendon regeneration using current tissue engineering techniques requires advanced biomaterials to satisfy both microstructural and mechanical criteria. In this study, a novel chitosan (CS)-based scaffold with asymmetric structure was fabricated using a self-deposition technique. The fabricated scaffolds were assessed with regard to the microstructural and mechanical demands of cell ingrowth and the prevention of peritendinous adhesion. In vitro studies showed that rat tendon stem/progenitor cells (TSPCs) seeded onto the CS scaffold displayed higher levels of tenogenic specific genes expression and protein production. Four and six weeks after the implantation of CS scaffolds on full-site Achilles tendon defects, in vivo tendon repair was evaluated by histology, immunohistochemistry, immunofluorescence, and mechanical measurements. The production of collagen I (COL1) and collagen III (COL3) demonstrated that the CS scaffolds were capable of inducing conspicuous tenogenic differentiation, higher tenomodulin (TNMD) production, and superior phenotypic maturity, compared with the empty defect group. The introduction of TSPCs into the CS scaffold resulted in a synergistic effect on tendon regeneration and yielded better-aligned collagen fibers with elongated, spindle-shaped cells. These findings indicated that the application of TSPC-seeded CS scaffolds would be a feasible approach for tendon repair. Statement of SignificanceThe poor healing capacity of injured tendons and inevitable peritendinous adhesion has made tendon regeneration a clinical priority. In this study, an asymmetric chitosan scaffold was developed to encapsulate rat tendon stem/progenitor cells (TSPCs), which could induce higher levels of tenogenic specific genes and protein expression. Remarkably, the introduction of TSPCs into the asymmetric chitosan scaffold generated a synergistic effect on in vivo tendon regeneration and lead to better-aligned collagen fibers compared with asymmetric chitosan scaffold alone. This work can provide new guidelines for the structure and property design of cell-seeded scaffolds for tendon regeneration.

Effect of scaffold morphology and cell co-culture on tenogenic differentiation of HADMSC on centrifugal melt electrospun poly (L‑lactic acid) fibrous meshes

Engineered tendon grafts offer a promising alternative for grafting during the reconstruction of complex tendon tears. The tissue-engineered tendon substitutes have the advantage of increased biosafety and the option to customize their biochemical and biophysical properties to promote tendon regeneration. In this study, we developed a novel centrifugal melt electrospinning (CME) technique, with the goal of optimizing the fabrication parameters to generate fibrous scaffolds for tendon tissue engineering. The effects of CME processing parameters, including rotational speed, voltage, and temperature, on fiber properties (i.e. orientation, mean diameter, and productivity) were systematically investigated. By using this solvent-free and environmentally friendly method, we fabricated both random and aligned poly (L-lactic acid) (PLLA) fibrous scaffolds with controllable mesh thickness. We also investigated and compared their morphology, surface hydrophilicity, and mechanical properties. We seeded human adipose derived mesenchymal stem cells (HADMSC) on various PLLA fibrous scaffolds and conditioned the constructs in tenogenic differentiation medium for up to 21 days, to investigate the effects of fiber alignment and scaffold thickness on cell behavior. Aligned fibrous scaffolds induced cell elongation and orientation through a contact guidance phenomenon and promoted HADMSC proliferation and differentiation towards tenocytes. At the early stage, thinner scaffolds were beneficial for HADMSC proliferation, but the scaffold thickness had no significant effects on cell proliferation for longer-term cell culture. We further co-seeded HADMSC and human umbilical vein endothelial cells (HUVEC) on aligned PLLA fibrous mats and determined how the vascularization affected HADMSC tenogenesis. We found that co-cultured HADMSC–HUVEC expressed more tendon-related markers on the aligned fibrous scaffold. The co-culture systems promoted in vitro HADMSC differentiation towards tenocytes. These aligned fibrous scaffolds fabricated by CME technique could potentially be utilized to repair and regenerate tendon defects and injuries with cell co-culture and controlled vascularization.

Preparation of collagen/polyurethane/knitted silk as a composite scaffold for tendon tissue engineering.

The main objective of this study was to prepare a hybrid three-dimensional scaffold that mimics natural tendon tissues. It has been found that a knitted silk shows good mechanical strength; however, cell growth on the bare silk is not desirable. Hence, electrospun collagen/polyurethane combination was used to cover knitted silk. A series of collagen and polyurethane solutions (4%-7% w/v) in aqueous acetic acid were prepared and electrospun. According to obtained scanning electron microscopy images from pure collagen and polyurethane nanofibers, concentration was set constant at 5% (w/v) for blend solutions of collagen/polyurethane. Afterward, blend solutions with the weight ratios of 75/25, 50/50 and 25/75 were electrospun. Scanning electron microscopy images demonstrated the smooth and uniform morphology for the optimized nanofibers. The least fibers diameter among three weight ratios was found for collagen/polyurethane (25/75) which was 100.86 ± 40 nm and therefore was selected to be electrospun on the knitted silk. Attenuated total reflectance-Fourier transform infrared spectra confirmed the chemical composition of obtained electrospun nanofibers on the knitted silk. Tensile test of the specimens including blend nanofiber, knitted silk and commercial tendon substitute examined and indicated that collagen/polyurethane-coated knitted silk has appropriate mechanical properties as a scaffold for tendon tissue engineering. Then, Alamar Blue assay of the L929 fibroblast cell line seeded on the prepared scaffolds demonstrated appropriate viability of the cells with a significant proliferation on the scaffold containing more collagen content. The results illustrate that the designed structure would be promising for being used as a temporary substitute for tendon repair.

Preparation and characterization of non-osteogenic compact bone scaffold for tendon tissue engineering

Preparation and characterization of non-osteogenic compact bone scaffold for tendon tissue engineering

Direct E-jet printing of three-dimensional fibrous scaffold for tendon tissue engineering.

Tissue engineering (TE) offers a promising strategy to restore diseased tendon tissue. However, a suitable scaffold for tendon TE has not been achieved with current fabrication techniques. Herein, we report the development of a novel electrohydrodynamic jet printing (E-jetting) for engineering 3D tendon scaffold with high porosity and orientated micrometer-size fibers. The E-jetted scaffold comprised tubular multilayered micrometer-size fibrous bundles, with interconnected spacing and geometric anisotropy along the longitudinal direction of the scaffold. Fiber diameter, stacking pattern, and interfiber distance have been observed to affect the structural stability of the scaffold, of which the enhanced mechanical strength can be obtained for scaffolds with thick fibers as the supporting layer. Human tenocytes showed a significant increase in cellular metabolism on the E-jetted scaffolds as compared to that on conventional electrospun scaffolds (2.7-, 2.8-, and 3.1-fold increase for 150, 300, and 600 µm interfiber distance, respectively; p < 0.05). Furthermore, the scaffolds provided structural support for human tenocytes to align with controlled orientation along the longitudinal direction of the scaffold, and promoted the expression of collagen type I. For the first time, E-jetting has been explored as a novel scaffolding approach for tendon TE, and offers a 3D fibrous scaffold to promote organized tissue reconstruction for potential tendon healing. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 616-627, 2017.

Preparation and Functional Characterization of Decellularized Bovine Tendon Scaffolds for Tendon Tissue Engineering

The study was conducted to develop a naturally derived tendon tissue engineered scaffold with the preservation of the native ultra-structure, tensile strength and biochemical composition of the tendon extracellular matrix. Decellularization was achieved by using two different protocols viz. repeated freeze and thaw technique and 2% SDS. In vitro evaluation of the graft was done by H&E staining, Masson-Trichome staining, Alcian blue staining, DNA quantification analysis and Scanning Electron Microscopy (SEM). The in vitro evaluation suggested that SDS was better than freeze and thaw technique in terms of effective decellularization. The cell removal was better in SDS group than freeze and thaw as depicted by H&E staining and DNA quantification. Also the structure and alignment of collagen fibers were preserved in SDS group. The intrinsic ultra structure of tendon tissue was well preserved based on scanning electron microscopy examination.

A novel electrospun-aligned nanoyarn-reinforced nanofibrous scaffold for tendon tissue engineering

An electrospun-aligned nanoyarn-reinforced nanofibrous scaffold (NRS) was developed for tendon tissue engineering to improve mechanical strength and cell infiltration. The novel scaffold composed of aligned nanoyarns and random nanofibers was fabricated via electrospinning using a two-collector system. The aim of the present study was to investigate three different types of electrospun scaffolds (random nanofibrous scaffold, aligned nanofibrous scaffold and NRS) based on silk fibroin (SF) and poly(l-lactide-co-caprolactone) blends. Morphological analysis demonstrated that the NRS composed of aligned nanoyarns and randomly distributed nanofibers formed a 3D microstructure with relatively large pore sizes and high porosity. Biocompatibility analysis revealed that bone marrow-derived mesenchymal stem cells exhibited a higher proliferation rate when cultured on the NRS compared with the other scaffolds. The mechanical testing results indicated that the tensile properties of the NRS were reinforced in the direction parallel to the nanoyarns and satisfied the mechanical requirements for tendon repair. In addition, cell infiltration was significantly enhanced on the NRS. In conclusion, with its improved porosity and appropriate mechanical properties, the developed NRS shows promise for tendon tissue engineering applications.

Freeze-Thaw Cycles Enhance Decellularization of Large Tendons

The use of decellularized tendon tissue as a scaffold for tendon tissue engineering provides great opportunities for future clinical and current research applications. The aim of this study was to assess the effect of repetitive freeze-thaw cycles and two different detergents, t-octyl-phenoxypolyethoxyethanol (Triton X-100) and sodium dodecyl sulfate (SDS), on decellularization effectiveness and cytocompatibility in large tendons. Freshly collected equine superficial and deep digital flexor tendons were subjected to decellularization according to four different protocols (1 and 2: freeze-thaw cycles combined with either Triton X-100 or SDS; 3 and 4: Triton X-100 or SDS). Decellularization effectiveness was assessed based on the reduction of vital cell counts, histologically visible nuclei, and DNA content. Transmission electron microscopy was performed to evaluate cellular and extracellular matrix integrity. Further, cytocompatibility of scaffolds that had been decellularized according to the protocols including freeze-thaw cycles (protocols 1 and 2) was assessed by seeding the scaffolds with superparamagnetic iron oxide labeled mesenchymal stromal cells and monitoring the cells histologically and by magnetic resonance imaging for two weeks. Decellularization was significantly more effective when using the protocols including freeze-thaw cycles, leaving only roughly 1% residual nuclei and 20% residual DNA, whereas samples that had not undergone additional freeze-thaw cycles contained roughly 20% residual nuclei and 40% residual DNA. No morphological extracellular matrix alterations due to decellularization could be observed. Scaffolds prepared by both protocols including freeze-thaw cycles were cytocompatible, but the cell distribution into the scaffold tended to be better in scaffolds that had been decellularized using freeze-thaw cycles combined with Triton X-100 instead of SDS.

Enhancement of tenogenic differentiation of human adipose stem cells by tendon-derived extracellular matrix

Mesenchymal stem cells (MSCs) have gained increasing research interest for their potential in improving healing and regeneration of injured tendon tissues. Developing functional three-dimensional (3D) scaffolds to promote MSC proliferation and differentiation is a critical requirement in tendon tissue engineering. Tendon extracellular matrix has been shown to maintain the tenogenic potential of tendon stem cells and stimulate tenogenesis of human adipose stem cells (hASCs) in 2D culture. This study aims at characterizing the biological composition of urea-extracted fraction of tendon ECM (tECM) and its tenogenic effect on hASCs cultured in a 3D collagen scaffold under uniaxial tension. The tECM obtained was cell-free and rich in ECM proteins. hASCs seeded in tECM-supplemented scaffold exhibited significantly increased proliferation and tenogenic differentiation. The presence of tECM also greatly suppressed the osteogenic differentiation of hASCs triggered by uniaxial tension. In addition, tECM-supplemented constructs displayed enhanced mechanical strength, accompanied by reduced expression and activity of MMPs in the seeded hASCs, indicating a regulatory activity of tECM in cell-mediated scaffold remodeling. These findings support the utility of tECM in creating bio-functional scaffolds for tendon tissue engineering.

Multilayered Electrospun Scaffolds for Tendon Tissue Engineering

Full-thickness rotator cuff tears are one of the most common causes of shoulder pain in people over the age of 65. High retear rates and poor functional outcomes are common after surgical repair, and currently available extracellular matrix scaffold patches have limited abilities to enhance new tendon formation. In this regard, tissue-engineered scaffolds may provide a means to improve repair of rotator cuff tears. Electrospinning provides a versatile method for creating nanofibrous scaffolds with controlled architectures, but several challenges remain in its application to tissue engineering, such as cell infiltration through the full thickness of the scaffold as well as control of cell growth and differentiation. Previous studies have shown that ligament-derived extracellular matrix may enhance differentiation toward a tendon or ligament phenotype by human adipose stem cells (hASCs). In this study, we investigated the use of tendon-derived extracellular matrix (TDM)-coated electrospun multilayered scaffolds compared to fibronectin (FN) or phosphate-buffered saline (PBS) coating for use in rotator cuff tendon tissue engineering. Multilayered poly(ɛ-caprolactone) scaffolds were prepared by sequentially collecting electrospun layers onto the surface of a grounded saline solution into a single scaffold. Scaffolds were then coated with TDM, FN, or PBS and seeded with hASCs. Scaffolds were maintained without exogenous growth factors for 28 days in culture and evaluated for protein content (by immunofluorescence and biochemical assay), markers of tendon differentiation, and tensile mechanical properties. The collagen content was greatest by day 28 in TDM-scaffolds. Gene expression of type I collagen, decorin, and tenascin C increased over time, with no effect of scaffold coating. Sulfated glycosaminoglycan and dsDNA contents increased over time in culture, but there was no effect of scaffold coating. The Young's modulus did not change over time, but yield strain increased with time in culture. Histology demonstrated cell infiltration through the full thickness of all scaffolds and immunofluorescence demonstrated greater expression of type I, but not type III collagen through the full thickness of the scaffold in TDM-scaffolds compared to other treatment groups. Together, these data suggest that nonaligned multilayered electrospun scaffolds permit tenogenic differentiation by hASCs and that TDM may promote some aspects of this differentiation.

Functional characterization of detergent-decellularized equine tendon extracellular matrix for tissue engineering applications.

Natural extracellular matrix provides a number of distinct advantages for engineering replacement orthopedic tissue due to its intrinsic functional properties. The goal of this study was to optimize a biologically derived scaffold for tendon tissue engineering using equine flexor digitorum superficialis tendons. We investigated changes in scaffold composition and ultrastructure in response to several mechanical, detergent and enzymatic decellularization protocols using microscopic techniques and a panel of biochemical assays to evaluate total protein, collagen, glycosaminoglycan, and deoxyribonucleic acid content. Biocompatibility was also assessed with static mesenchymal stem cell (MSC) culture. Implementation of a combination of freeze/thaw cycles, incubation in 2% sodium dodecyl sulfate (SDS), trypsinization, treatment with DNase-I, and ethanol sterilization produced a non-cytotoxic biomaterial free of appreciable residual cellular debris with no significant modification of biomechanical properties. These decellularized tendon scaffolds (DTS) are suitable for complex tissue engineering applications, as they provide a clean slate for cell culture while maintaining native three-dimensional architecture.