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Enhancing mechanical properties of tissue-engineered constructs via lysyl oxidase crosslinking activity.

A number of strategies have been investigated to enhance the mechanical stability of engineered tissues. In this study, we utilized lysyl oxidase (LO) to enzymatically crosslink extracellular matrix (ECM) proteins, particularly collagen and elastin, to enhance the mechanical integrity of the ECM and thereby impart mechanical strength to the engineered tissue. Vascular smooth muscle cells (VSMCs) were liposomally transfected with the LO gene. Both Northern and Western analyses confirmed increased LO expression. Increased LO activity was demonstrated using a fluorescent enzyme substrate assay and by observation of the presence of increased levels of desmosine, a product of LO crosslinking, in the ECM. The mechanical effects of altered crosslink densities within tissue-engineered constructs were demonstrated in a VSMC-populated collagen gel model. When smooth muscle cells transfected with lysyl oxidase were seeded in collagen gels, the tensile strength and elastic modulus in these constructs increased by approximately two-fold compared to constructs seeded with mock-transfected VSMCs. Also, desmosine levels in the LO-populated collagen gels were higher than they were in mock-seeded gels, as demonstrated via immunohistochemical staining. Compositional analysis of the ECM deposited by the transformed cells showed similar collagen and elastin levels, and cell proliferation rates were similar as well, thus attributing increased mechanical properties to ECM crosslinking.

Animals↗

Quantitative analysis of three-dimensional fluid flow in rotating bioreactors for tissue engineering.

Tissue engineering has emerged as a viable alternative to the problem of organ and tissue shortage. Our laboratory has developed matrices for bone tissue engineering based on sintered spherical particles and, using bioreactor technology, has demonstrated the ability to produce highly mineralized matrices in vitro. In this study, porous microcapsule scaffolds were developed for bone tissue engineering in the high aspect ratio vessel rotating bioreactor. The motion of individual microcapsules as well as scaffolds in the bioreactor were studied by numerical simulation and in situ imaging analysis. Results show that spherical microcapsules with density less than the surrounding fluid exhibited two motions: (1) a periodic circular orbit with tangential speed equal to the free fall speed of the particle, and (2) an inward radial migration of the circular orbit toward the center of the bioreactor vessel. Lighter-than-water scaffolds were fabricated by sintering poly(lactic-co-glycolic acid) hollow microcarriers with diameter from 500 to 860 microm into a fixed three-dimensional geometry with approximately 30% pore volume and 180 to 190 microm median pore size. Scaffolds were fabricated with aggregate densities ranging from 0.65 g/mL and 0.99 g/mL by appropriate combinations of hollow and solid microcarriers within the scaffold. Scaffold velocity in the bioreactor for the above range of densities was accurately predicted by numerical simulation and ranged from 100 mm/s to 3 mm/s. Maximum shear stress estimation due to media flow over the exterior of the scaffold ranged from 0.3 N/m(2) to 0.006 N/m(2). Internal perfusion velocity through scaffolds also was calculated and ranged from 13 mm/s to 0.2 mm/s. Estimates of maximum interior shear stress ranged from 0.03 to 0.0007 N/m(2). These analytical methods provide an excellent vehicle for the study of bone tissue synthesis in three-dimensional culture with fluid flow.

Bioreactors↗

The use of a novel PLGA fiber/collagen composite web as a scaffold for engineering of articular cartilage tissue with adjustable thickness.

It has been a great challenge to make the thickness of engineered cartilage adjustable to cover the range of both partial-thickness and full-thickness articular cartilage defects. We developed a novel kind of composite web scaffold that could be used for tissue enginnering of articular cartilage with the thickness adjustable between 200 microm and 8 mm. The composite web showed a unique structure having web-like collagen microsponges formed in the openings of a mechanically strong knitted mesh of poly(lactic-co-glycolic acid). The knitted mesh served as a skeleton reinforcing the composite web, while the web-like collagen microsponges facilitated cell seeding, cell distribution, and tissue formation. Bovine chondrocytes cultured in the composite web showed a spatially even distribution, maintained their natural morphology, and produced cartilaginous extracellular matrices such as type II collagen and aggrecan. The thickness of the implant can be simply adjusted by laminating or rolling the web sheets. Not only did the histological structure of the engineered cartilage patches match the bovine native articular cartilage, but also their dynamic complex modulus, structural stiffness, and phase lag reached 37.8, 57.0, and 86.3% of those of native bovine articular cartilage, respectively. The composite web could be an important scaffold for tissue engineering.

Aggrecans↗

Characterization of statically loaded tissue-engineered mitral valve chordae tendineae.

Chordae tendineae are essential to the proper function of the mitral valve. Native chordae contain a dense collagenous core and an outer elastin sheath. We have been using the principle of directed collagen gel shrinkage to fabricate tissue-engineered mitral valve chordae. Because the microstructure of biologic tissues determines their mechanical behavior, the morphology of collagen and elastin in tissue-engineered chordae should mimic that of native chordae. The objective of this study, therefore, was to examine the morphology of our tissue-engineered constructs in comparison to native chordae. A collagen-cell suspension was cast into silicon rubber wells with microporous anchors at the ends and cultured in an incubator. The anchors allowed shrinkage to occur only transverse to the long axis of the wells, thus creating highly aligned collagen fibril constructs. The collagen constructs were cultured for 8 weeks and characterized mechanically, histologically, and biochemically at different culture time points. Histologic sections showed that in all mature constructs collagen fibers were oriented parallel to the long axis of the constructs. At the edge of the tissue collagen fibers were in general straight, whereas in the middle of the tissue they were wavy. Transmission electron microscopy showed a progressive increase in the density and longitudinal orientation of collagen fibrils with culture time. Light and scanning electron microscopy showed the presence of an elastin sheath around the collagen core. Immunostaining demonstrated that smooth muscle cells differentiate during tissue development and TUNEL assay showed that cells in the interior of the constructs undergo apoptosis. This study has demonstrated that collagen-cell constructs, with material properties and microstructure similar to native mitral valve chordae, can be developed using static culture.

Animals↗

Thermoreversible hydrogel scaffolds for articular cartilage engineering.

Articular cartilage has limited potential for repair. Current clinical treatments for articular cartilage damage often result in fibrocartilage and are associated with joint pain and stiffness. To address these concerns, researchers have turned to the engineering of cartilage grafts. Tissue engineering, an emerging field for the functional restoration of articular cartilage and other tissues, is based on the utilization of morphogens, scaffolds, and responding progenitor/stem cells. Because articular cartilage is a water-laden tissue and contains within its matrix hydrophilic proteoglycans, an engineered cartilage graft may be based on synthetic hydrogels to mimic these properties. To this end, we have developed a polymer system based on the hydrophilic copolymer poly(propylene fumarate-co-ethylene glycol) [P(PF-co-EG)]. Solutions of this polymer are liquid below 25 degrees C and gel above 35 degrees C, allowing an aqueous solution containing cells at room temperature to form a hydrogel with encapsulated cells at physiological body temperature. The objective of this work was to determine the effects of the hydrogel components on the phenotype of encapsulated chondrocytes. Bovine articular chondrocytes were used as an experimental model. Results demonstrated that the components required for hydrogel fabrication did not significantly reduce the proteoglycan synthesis of chondrocytes, a phenotypic marker of chondrocyte function. In addition, chondrocyte viability, proteoglycan synthesis, and type II collagen synthesis within P(PF-co-EG) hydrogels were investigated. The addition of bone morphogenetic protein-7 increased chondrocyte proliferation with the P(PF-co-EG) hydrogels, but did not increase proteoglycan synthesis by the chondrocytes. These results indicate that the temperature-responsive P(PF-co-EG) hydrogels are suitable for chondrocyte delivery for articular cartilage repair.

Animals↗

Fabrication and evaluation of porous 2,3-dialdehydecellulose membrane as a potential biodegradable tissue-engineering scaffold.

A simple, novel method to produce porous 2,3-dialdehydecellulose (DAC) membranes as a potential tissue-engineering scaffold has been developed from methylolcellulose by the simultaneous water-induced phase separation and sodium chloride salt leaching techniques, followed by oxidation with sodium periodate in water. Membrane pores increased in size with increasing weight or particle size of the sodium chloride salt. The porosity of the membrane was not affected by the salt particle size, but it increased with an increase in the salt weight to 60%. At higher salt weight percentages, no significant change in the membrane porosity was observed. The oxidation step had no effect on the membrane porosity or pore size. All membranes with a porosity value ranging between 87 and 93% showed interconnected porous structures. The use of these membranes as a potential tissue-engineering scaffold was evaluated with the use of human neonatal skin fibroblast cells. Confocal microscopy showed cell attachment and spreading on these membranes. Immunohistochemical tests revealed the presence of collagen type III and fibronectin, indicating that the cells were viable and formed the extracellular matrix. In conclusion, the DAC membrane supports cell adhesion and proliferation and hence shows potential to be used as a tissue-engineering scaffold.

Absorbable Implants↗

A new biodegradable polyester elastomer for cartilage tissue engineering.

The objective of this study is to assess whether a new biodegradable elastomer, poly(1,8-octanediol citrate) (POC), would be a suitable material to engineer elastomeric scaffolds for cartilage tissue engineering. Porous POC scaffolds were prepared via the salt-leaching method and initially assessed for their ability to rapidly recover from compressive deformation (% recovery ratio). Controls consisted of scaffolds made from other materials commonly used in cartilage tissue engineering, including 2% agarose, 4% alginate, non woven poly(glycolic acid) (PGA) meshes, and non woven poly(L-lactide-co-glycolide) (PLGA) meshes. Articular chondrocytes from bovine knee were isolated and seeded onto porous disk-shaped POC scaffolds, which were subsequently cultured in vitro for up to 28 days. POC scaffolds completely recover from compressive deformation, and the stress-strain curve is typical of an elastomer (recovery ratio>98%). Agarose gel (2%) scaffolds broke during the compression test. The recovery ratio of 4% alginate gel scaffolds, PLLA, and PGA were 72, 85, and 88%, respectively. The Young's modulus of POC-chondrocyte constructs and cell-free POC scaffolds cultured for 28 days were 12.02+/-2.26 kPa and 3.27+/-0.72 kPa, respectively. After 28 days of culture, the recovery ratio of POC-chondrocyte constructs and cell-free POC scaffolds were 93% and 99%, respectively. The glycosaminoglycan (GAG) and collagen content at day 28 was 36% and 26% of that found in bovine knee cartilage explants. Histology/immunohistochemistry evaluations confirm that chondrocytes were able to attach to the pore walls within the scaffold, maintain cell phenotype, and form a cartilaginous tissue during the 28 days of culture.

Absorbable Implants↗

Thermoreversible laminin-functionalized hydrogel for neural tissue engineering.

Traumatic injury to the central nervous system (CNS) triggers cell death and deafferentation, which may activate a cascade of cellular and network disturbances. These events often result in the formation of irregularly shaped lesions comprised of necrotic tissue and/or a fluid-filled cavity. Tissue engineering represents a promising treatment strategy for the injured neural tissue. To facilitate minimally invasive delivery of a tissue engineered system, a thermoreversible polymer is an attractive scaffold candidate. We have developed a bioactive scaffold for neural tissue engineering by tethering laminin-1 (LN) to methylcellulose (MC), a thermoresponsive hydrogel. The base MC chain was oxidized via sodium m-periodate to increase MC tethering capacity. Protein immobilization was facilitated by a Schiff base reaction between primary amine groups on LN and the carbonyl groups of the oxidized MC chain. Immunoassays demonstrated tethering of LN at 1.6 +/- 0.5 ng of LN per milligram of MC. Rheological measurements for different MC-LN constructs indicated MC composition- and MC treatment-dependent effects on solution-gelation transition temperature. Cellular assays with primary rat cortical neurons demonstrated enhanced cell adhesion and viability on LN-functionalized MC when compared with base and oxidized MC. This bioadhesive thermoresponsive scaffold may provide a robust delivery vehicle to injured CNS tissue for neural cell transplantation strategies.

Animals↗

Radial artery as an autologous cell source for valvular tissue engineering efforts.

To create a viable tissue-engineered aortic valve, it is important to identify suitable autologous cell sources that may be seeded onto a biocompatible scaffold. This study focused on the radial artery (RA) as one possible source, investigated optimal culture conditions, and determined the usefulness of small intestinal submucosa (SIS) as a scaffold for tissue-engineering. Porcine RA cells were cultured on either two-dimensional (2D) 100-mm dishes or three-dimensional (3D) 1-cm(2) SIS sheets, producing cell-scaffold composites (CSCs). Both 2D and 3D cultures were maintained in either Medium 199 (M199) or endothelial growth media (EGM) to determine optimal growth conditions. Cellular phenotype and matrix metalloproteinase (MMP) profiles were determined by immunoblotting of cell lysates and zymography of conditioned media, respectively. Cellular invasion was analyzed immunohistochemically on CSC tissue sections. We show that the RA contains phenotypes consistent with those found in the normal aortic valve. EGM, compared with M199, promotes the invasion and remodeling of SIS by RA cells, which is crucial in the process of replacing the foreign tissue scaffold prior to implantation. To our knowledge, this is the first study to show that the RA is a suitable source for the generation of a tissue-engineered valve.

Animals↗

Surface adsorption of DNA to tissue engineering scaffolds for efficient gene delivery.

Gene delivery from tissue engineering scaffolds has potential to promote localized transgene expression that can induce the formation of functional tissues. Substrate-mediated delivery, an alternative delivery strategy to sustained release, is based on immobilization of DNA complexes to the polymer surface for subsequent delivery to cells cultured on the substrate. We investigate polyethylenimine (PEI)/DNA complex immobilization and subsequent cellular transfection on tissue engineering scaffolds fabricated from poly(lactide-co-glycolide) (PLG). The properties of the substrate and the complex affect both immobilization and cellular transfection. PLG promotes binding of PEI/DNA complexes, with percent bound independent of the N/P ratio or the DNA dosage. The levels of transgene expression are similar to or greater than control studies based on bolus DNA delivery, with orders of magnitude less DNA. Immobilization also homogeneously distributes the DNA throughout the scaffold, resulting in large numbers of transfected cells (>60%) at low surface quantities (<50 ng). Importantly, this approach can be employed to transfect cells throughout a three-dimensional scaffold. Tissue engineering scaffolds that are prefabricated into various shapes from a range of materials could potentially employ this strategy for numerous applications.

Adsorption↗

Dynamic contrast-enhanced MRI to quantify VEGF-enhanced tissue-engineered bladder graft neovascularization: pilot study.

Tissue engineered organs require an immediately perfused vascular tree. Currently, neovascularization assessment requires animal sacrifice and graft harvest. In this pilot study we assess whether neovascularization of an engineered urinary bladder construct is enhanced with vascular-derived endothelial growth factor (VEGF) and assessable non-invasively with dynamic contrast-enhanced MRI (DCE-MRI). Rabbit bladder acellular matrix was hybridized with hyaluronic acid (ACM-HA), fortified with one of three concentrations (0 ng, 10 ng, or 20 ng per gram of tissue) of vascular-derived endothelial growth factor (VEGF), and grafted onto bladders in nine rabbits (3 per VEGF concentration). At 1, 2 and 3 weeks, one rabbit from each VEGF group underwent DCE-MRI and graft harvest. Microvasculature was quantified with scrial optical transverse sectioning of CD31 stained whole mounts using PCl software. Masson trichrome and H&E staining were used to assess cellular proliferation and fibrosis. There was a significant difference in mean microvascular area (MVA) between the 10 and 20 ng VEGF groups (230187 vs 477894 microm2, P=0.014) but not between the 0 and 10 ng groups (210497 microm2, P=0.21). Gadolinium uptake increased with MVA and correlated with it on linear regression analysis (Pearson's correlation coefficient r=0.71). At three weeks, stromal cellularity was greatest, and fibrosis was least, in the high VEGF group. This preliminary work demonstrates improved neovascularization of bladder constructs with VEGF fortification of ACM-HA and the feasibility of MRI as a non-invasive method to assume angiogenesis in tissue engineered constructs.

Animals↗

Porous chitosan tubular scaffolds with knitted outer wall and controllable inner structure for nerve tissue engineering.

In this study, a novel method was developed to create porous tubular scaffolds with desirable mechanical properties and controllable inner structure from chitosan, for nerve tissue engineering. Chitosan fiber-based yarns were first used to create porous hollow tubes, which served as the outer wall of the scaffolds, through an industrial knitting process. Then, an innovative molding technique was developed and used to produce inner matrices with multiple axially oriented macrochannels and radially interconnected micropores. Acupuncture needles were used as mandrels during molding to improve the safety and controllability of the process. In vitro characterization demonstrated that the scaffolds possessed suitable mechanical strength, porosity, swelling, and biodegradability for applications in nerve tissue engineering. In vitro cell culture experiments showed that differentiated Neuro-2a cells grew along the oriented macrochannels and the interconnected micropores were beneficial for nutrient diffusion and cell ingrowth to the scaffold's interior. Collectively, the well-defined architectural features in addition to the desirable mechanical and biological properties of the scaffolds make them promising for nerve tissue engineering.

Animals↗

Electrospun fibrinogen: feasibility as a tissue engineering scaffold in a rat cell culture model.

Fibrinogen has a well-established tissue engineering track record because of its ability to induce improved cellular interaction and scaffold remodeling compared to synthetic scaffolds. While the feasibility of electrospinning fibrinogen scaffolds of submicron diameter fibers and their mechanical properties have been demonstrated, in vitro cellular interaction has not yet been evaluated. The goal of this study was to demonstrate, based on cellular interaction and scaffold remodeling, that electrospun fibrinogen can be used successfully as a tissue engineering scaffold. Electrospun fibrinogen scaffolds were disinfected, seeded with neonatal rat cardiac fibroblasts, and cultured for 2, 7, and 14 days. Cultures were treated to regulate scaffold degradation by either supplementing serum-containing media with aprotinin or crosslinking the scaffolds with glutaraldehyde vapor. Biocompatibility was assessed through a WST-1 cell proliferation assay. Postculture scaffolds were evaluated by scanning electron microscopy and histology. Cell culture demonstrated that fibroblasts readily migrate into and remodel electrospun fibrinogen scaffolds with deposition of native collagen. Supplementation of culture media with different concentrations of aprotinin-modulated scaffold degradation in a predictable fashion, but glutaraldehyde vapor fixation was less reliable. Based on the observed cellular interactions, there is tremendous potential for electrospun fibrinogen as a tissue engineering scaffold.

Animals↗

Fast and efficient screening system for new biomaterials in tissue engineering: a model for peripheral nerve regeneration.

The use of three-dimensional biodegradable matrices is one major issue in tissue engineering. Numerous materials, fabrication techniques, and modifications have been used and tested in different areas of tissue engineering recently. But nevertheless, technology is far from being optimized and optimal constructs with bioidentical and mechanical properties have not been described in the literature so far. Hence, there is great demand of new suitable biomaterials for tissue engineering applications. In this study, a fast and efficient screening system for initial testing of biomaterials for cell culture application was developed. The set up for the screening system and the decision criteria applied for the determination of suitability of new materials are presented. Hep-G2 and PC-12 cells were seeded onto different matrices and cultured over a period of 2 weeks. The viability of the cells was monitored via the MTT assay. Cell spreading was investigated by DAPI-staining of cell nuclei. Furthermore, the adhesion of the cells on the different matrices was examined by counting the number of attached cells. With these general assays a classification of materials is possible with regard to their suitability. Optimal cell models must be chosen for the defined applications and at least two cell lines are necessary for a differentiating interpretation.

Animals↗

Molecular diffusion in tissue-engineered cartilage constructs: effects of scaffold material, time, and culture conditions.

Diffusion is likely to be the primary mechanism for macromolecular transport in tissue-engineered cartilage, and providing an adequate nutrient supply via diffusion may be necessary for cell proliferation and extracellular matrix production. The goal of this study was to measure the diffusivity of tissue-engineered cartilage constructs as a function of scaffold material, culture conditions, and time in culture. Diffusion coefficients of four different-sized fluorescent dextrans were measured by fluorescence recovery after photobleaching in tissue-engineered cartilage constructs seeded with human adipose-derived stem cells or acellular constructs on scaffolds of alginate, agarose, gelatin, or fibrin that were cultured for 1 or 28 days in either chondrogenic or control conditions. Diffusivities in the constructs were much greater than those of native cartilage. The diffusivity of acellular constructs increased 62% from Day 1 to Day 28, whereas diffusivity of cellular constructs decreased 42% and 27% in chondrogenic and control cultures, respectively. The decrease in diffusivity in cellular constructs is likely due to new matrix synthesis, which may be enhanced with chondrogenic media, and matrix contraction by the cells in the fibrin and gelatin scaffolds. The increase in diffusivity in the acellular constructs is probably due to scaffold degradation and swelling.

Adipocytes↗

Chitosan scaffolds for in vitro buffalo embryonic stem-like cell culture: an approach to tissue engineering.

Three-dimensional (3D) porous chitosan scaffolds are attractive candidates for tissue engineering applications. Chitosan scaffolds of 70, 88, and 95% degree of deacetylation (% DD) with the same molecular weight were developed and their properties with buffalo embryonic stem-like (ES-like) cells were investigated in vitro. Scaffolds were fabricated by freezing and lyophilization. They showed open pore structure with interconnecting pores under scanning electron microscopy (SEM). Higher % DD chitosan scaffolds had greater mechanical strength, slower degradation rate, lower water uptake ability, but similar water retention ability, when compared to lower % DD chitosan. As a strategy to tissue engineering, buffalo ES-like cells were cultured on scaffolds for 28 days. It appeared that chitosan was cytocompatible and cells proliferated well on 88 and 95% DD scaffolds. In addition, the buffalo ES-like cells maintained their pluripotency during the culture period. Furthermore, the SEM and histological study showed that the polygonal buffalo ES-like cells proliferated well and attached to the pores. This study proved that 3D biodegradable highly deacetylated chitosan scaffolds are promising candidates for ES-like cell based tissue engineering and this chitosan scaffold and ES cell based system can be used as in vitro model for subsequent clinical applications.

Animals↗

Tissue-engineered intervertebral disc and chondrogenesis using human nucleus pulposus regulated through TGF-beta1 in platelet-rich plasma.

Human intervertebral disc (IVD) degeneration often initiated from the human nucleus pulposus (hNP) with aging leading to IVD destruction and extracellular matrix (ECM) depletion. Previously, we have successfully employed transforming growth factor-beta1 (TGF-beta1) to promote chondrogenesis of mesenchymal progenitor cells (MPCs) and immortalized human mesenchymal stem cells. In this study, we examine the role of TGF-beta1 in platelet-rich plasma (PRP) on disc regeneration, including proliferation, redifferentiation, and the reconstitution of tissue-engineered NP. hNP cells were isolated from volunteers with different ages and cultured in the presence of PRP. We found that the most effective concentration for hNP proliferation was 1 ng/ml TGF-beta1 in PRP, which was further applied in the following experiments. hNP cell proliferation in all age groups were increased time-dependently by PRP and cell morphologies showed aggregation. The mRNA of Sox9, type II collagen, and aggrecan were all significantly upregulated by PRP through RT-PCR. Glycosaminoglycan (GAG) accumulation reached the highest value at day 7 and continued to day 9 culture. PRP promoted NP regeneration via the Smad pathway was also determined and highly activated p-Smad2/3 at 30 min and continuously sustained to 120 min. Immunostaining of type II collagen indicates that PRP participates in chondrogenesis of tissue-engineered NP with collagen scaffolds. We concluded that growth factors in PRP can effectively react as a growth factor cocktail to induce hNP proliferation and differentiation, and also promote tissue-engineered NP formation. These findings are the first to demonstrate that PRP might be a therapeutic candidate for prevention of disc degeneration.

Adult↗

Gene therapy and tissue engineering for sports medicine.

Sports injuries usually involve tissues that display a limited capacity for healing. The treatment of sports injuries has improved over the past 10 to 20 years through sophisticated rehabilitation programs, novel operative techniques, and advances in the field of biomechanical research. Despite this considerable progress, no optimal solution has been found for treatment of various sports-related injuries, including muscle injuries, ligament and tendon ruptures, central meniscal tears, cartilage lesions, and delayed bone fracture healing. New biological approaches focus on the treatment of these injuries with growth factors to stimulate and hasten the healing process. Gene therapy using the transfer of defined genes encoding therapeutic proteins represents a promising way to efficiently deliver suitable growth factors into the injured tissue. Tissue engineering, which may eventually be combined with gene therapy, may potentially result in the creation of tissues or scaffolds for regeneration of tissue defects following trauma. In this article we will discuss why gene therapy and tissue engineering are becoming increasingly important in modern orthopaedic sports medicine practice. We then will review recent research achievements in the area of gene therapy and tissue engineering for sports-related injuries, and highlight the potential clinical applications of this technology in the treatment of patients with musculoskeletal problems following sports-related injuries.

Athletic Injuries↗