The mechanical properties of the human supraspinatus tendon (SST) are highly heterogeneous and ma... more The mechanical properties of the human supraspinatus tendon (SST) are highly heterogeneous and may reflect an important adaptive response to its complex, multiaxial loading environment. However, these functional properties are associated with a location-dependent structure and composition that have not been fully elucidated. Therefore, the objective of this study was to determine the concentrations of types I, II and III collagen in 6 distinct regions of the SST and compare changes in collagen concentration across regions with local changes in mechanical properties. We hypothesized that type I collagen content would be high throughout the tendon, type II collagen would be restricted to regions of compressive loading and type III collagen content would be high in regions associated with damage. We further hypothesized that regions of high type III collagen content would correspond to regions with low tensile modulus and a low degree of collagen alignment. Although type III collagen content was not significantly higher regions that are frequently damaged, all other hypotheses were supported by our results. In particular, type II collagen content was highest near the insertion while type III collagen was inversely correlated with tendon modulus and collagen alignment. The measured increase in type II collagen under the coracoacromial arch provides evidence of adaptation to compressive loading in the SST. Moreover, the structure-function relationship between type III collagen content and tendon mechanics established in this study demonstrates a mechanism for altered mechanical properties in pathological tendons and provides a guideline for identifying therapeutic targets and pathology-specific biomarkers.
Adult articular cartilage exhibits little capacity for intrinsic repair, and thus even minor inju... more Adult articular cartilage exhibits little capacity for intrinsic repair, and thus even minor injuries or lesions may lead to progressive damage and osteoarthritic joint degeneration, resulting in significant pain and disability. While there have been numerous attempts to develop tissue-engineered grafts or patches to repair focal chondral and osteochondral defects, there remain significant challenges in the clinical application of cell-based therapies for cartilage repair. This paper reviews the current state of cartilage tissue engineering with respect to different cell sources and their potential genetic modification, biomaterial scaffolds and growth factors, as well as preclinical testing in various animal models. This is not intended as a systematic review, rather an opinion of where the field is moving in light of current literature. While significant advances have been made in recent years, the complexity of this problem suggests that a multidisciplinary approach -combining a clinical perspective with expertise in cell biology, biomechanics, biomaterials science and high-throughput analysis will likely be necessary to address the challenge of developing functional cartilage replacements. With this approach we are more likely to realise the clinical goal of treating both focal defects and even large-scale osteoarthritic degenerative changes in the joint.
Cartilage tissue engineering is emerging as a promising treatment for osteoarthritis, and the fie... more Cartilage tissue engineering is emerging as a promising treatment for osteoarthritis, and the field has progressed toward utilizing large animal models for proof of concept and preclinical studies. Mechanical testing of the regenerative tissue is an essential outcome for functional evaluation. However, testing modalities and constitutive frameworks used to evaluate in vitro grown samples differ substantially from those used to evaluate in vivo derived samples. To address this, we developed finite element (FE) models (using FEBio) of unconfined compression and indentation testing, modalities commonly used for such samples. We determined the model sensitivity to tissue radius and subchondral bone modulus, as well as its ability to estimate material parameters using the built-in parameter optimization tool in FEBio. We then sequentially tested agarose gels of 4%, 6%, 8%, and 10% weight/weight using a custom indentation platform, followed by unconfined compression. Similarly, we evaluated the ability of the model to generate material parameters for living constructs by evaluating engineered cartilage. Juvenile bovine mesenchymal stem cells were seeded (2 • 10 7 cells/mL) in 1% weight/volume hyaluronic acid hydrogels and cultured in a chondrogenic medium for 3, 6, and 9 weeks. Samples were planed and tested sequentially in indentation and unconfined compression. The model successfully completed parameter optimization routines for each testing modality for both acellular and cell-based constructs. Traditional outcome measures and the FE-derived outcomes showed significant changes in material properties during the maturation of engineered cartilage tissue, capturing dynamic changes in functional tissue mechanics. These outcomes were significantly correlated with one another, establishing this FE modeling approach as a singular method for the evaluation of functional engineered and native tissue regeneration, both in vitro and in vivo.
Objective: Many approaches are being taken to generate cartilage replacement materials. The goal ... more Objective: Many approaches are being taken to generate cartilage replacement materials. The goal of this study was to use a self-aggregating suspension culture model of chondrocytes with mechanical preconditioning. Design: Our model differs from others in that it is based on a scaffold-less, self-aggregating culture model that produces a cartilage tissue analog that has been shown to share many similarities with the natural cartilage phenotype. Owing to the known loaded environment under which chondrocytes function in vivo, we hypothesized that applying force to the suspension culture-derived chondrocyte biomass would improve its cartilage-like characteristics and provide a new model for engineering cartilage tissue analogs. Results: In this study, we used a specialized hydrostatic pressure bioreactor system to apply mechanical forces during the growth phase to improve biochemical and biophysical properties of the biomaterial formed. We demonstrated that using this high-density suspension culture, a biomaterial more consistent with the hyaline cartilage phenotype was produced without any foreign material added. Unpassaged chondrocytes responded to a physiologically relevant hydrostatic load by significantly increasing gene expression of critical cartilage molecule collagen and aggrecan along with other cartilage relevant genes, CD44, perlecan, decorin, COMP, and iNOS. Conclusions: This study describes a self-aggregating bioreactor model without foreign material or scaffold in which chondrocytes form a cartilage tissue analog with many features similar to native cartilage. This study represents a promising scaffold-less, methodological advancement in cartilage tissue engineering with potential translational applications to cartilage repair.
Background: Post traumatic osteoarthritis is a disabling condition impacting mostly young and act... more Background: Post traumatic osteoarthritis is a disabling condition impacting mostly young and active population. In the present study we investigated the impact of intra-articular sprifermin, a recombinant truncated broblast growth factor 18, on the outcome of microfracture treatment, a widely used surgical technique to enhance cartilage healing at the site of injury. Methods: For the purpose if this study, we created a cartilage defect and performed microfracture treatment in fetlock joints of 18 horses, treated joints with one of three doses of sprifermin (10, 30, or 100 mg) or with saline, hyaluronan (HA), and evaluated animals functional and structural outcomes over 24 weeks. For primary outcome measures we performed histological evaluations and performed gene expression analysis of aggrecan, collagen types I and II, and cartilage oligomeric matrix protein (COMP) in three regions of interest. As secondary outcome measures we examined animal's lameness, performed arthroscopic, radiographic, and CT scan imaging, and gross morphology assessment. Results: We detected the highest treatment bene t following 100 mg sprifermin treatment. Overall histological assessment showed an improvement in the kissing region and expression of constitutive genes showed a concentration dependent enhancement, especially in the peri-lesion area. We detected a signi cant improvement in lameness scores, arthroscopic evaluations, radiography, and CT scans following sprifermin treatment when results from three dose-treatment groups were combined. Conclusion: Our results demonstrated for the rst time, an enhancement in outcomes of the microfracture treatment following sprifermin treatment, suggesting a cartilage regenerative role and a potential bene t of sprifermin treatment in early cartilage injuries.
The repair of focal cartilage defects remains one of the foremost issues in the field of orthopae... more The repair of focal cartilage defects remains one of the foremost issues in the field of orthopaedics. Chondral defects may arise from a variety of joint pathologies and left untreated, will likely progress to osteoarthritis. Current repair techniques, such as microfracture, result in short-term clinical improvements but have poor long-term outcomes. Emerging scaffold-based repair strategies have reported superior outcomes compared to microfracture and motivate the development of new biomaterials for this purpose. In this study, unique composite implants consisting of a base porous reinforcing component (woven poly(ε-caprolactone)) infiltrated with 1 of 2 hydrogels (self-assembling peptide or thermo-gelling hyaluronan) or bone marrow aspirate were evaluated. The objective was to evaluate cartilage repair with composite scaffold treatment compared to the current standard of care (microfracture) in a translationally relevant large animal model, the Yucatan minipig. While many cartilage-repair studies have shown some success in vivo, most are short term and not clinically relevant. Informed by promising 6-week findings, a 12-month study was carried out and those results are presented here. To aid in comparisons across platforms, several structural and functionally relevant outcome measures were performed. Despite positive early findings, the long-term results indicated less than optimal structural and mechanical results with respect to cartilage repair, with all treatment groups performing worse than the standard of care. This study is important in that it brings much needed attention to the importance of performing translationally relevant long-term studies in an appropriate animal model when developing new clinical cartilage repair approaches.
Delivery of biofactors in a precise and controlled fashion remains a clinical challenge. Stimulir... more Delivery of biofactors in a precise and controlled fashion remains a clinical challenge. Stimuliresponsive delivery systems can facilitate 'on-demand' release of therapeutics in response to a variety of physiologic triggering mechanisms (e.g. pH, temperature). However, few systems to date have taken advantage of mechanical inputs from the microenvironment to initiate drug release. Here, we developed mechanically-activated microcapsules (MAMCs) that are designed to deliver therapeutics in an on-demand fashion in response to the mechanically loaded environment of regenerating musculoskeletal tissues, with the ultimate goal of furthering tissue repair. To establish a suite of microcapsules with different thresholds for mechano-activation, we first manipulated MAMC physical dimensions and composition, and evaluated their mechano-response under both direct 2D compression and in 3D matrices mimicking the extracellular matrix properties and dynamic loading environment of regenerating tissue. To demonstrate the feasibility of this delivery system, we used an engineered cartilage model to test the efficacy of mechanically-instigated release of TGF-β3 on the chondrogenesis of mesenchymal stem cells. These data establish a novel platform by which to tune the release of therapeutics and/or
Mucopolysaccharidosis (MPS) VII is a lysosomal storage disorder characterized by deficient βglucu... more Mucopolysaccharidosis (MPS) VII is a lysosomal storage disorder characterized by deficient βglucuronidase activity, which leads to the accumulation of incompletely degraded glycosaminoglycans (GAGs). MPS VII patients present with severe skeletal abnormalities, which are particularly prevalent in the spine. Incomplete cartilage-to-bone conversion in MPS VII vertebrae during postnatal development is associated with progressive spinal deformity and spinal cord compression. The objectives of this study were to determine the earliest postnatal developmental stage at which vertebral bone disease manifests in MPS VII and to identify the underlying cellular basis of impaired cartilage-to-bone conversion, using the naturally-occurring canine model. Control and MPS VII dogs were euthanized at 9 and 14 days-of-age, and vertebral secondary ossification centers analyzed using micro-computed tomography, histology, qPCR, and protein immunoblotting. Imaging studies and mRNA analysis of bone formation markers established that secondary ossification commences between 9 and 14 days in control animals, but not in MPS VII animals. mRNA analysis of differentiation markers revealed that MPS VII epiphyseal *
Background-The concept of modulating spinal growth to correct scoliosis is intriguing, and this s... more Background-The concept of modulating spinal growth to correct scoliosis is intriguing, and this study proposes a new model. Inhibition of vertebral growth on the convex side of a curve would allow continued normal growth on the concave side to correct the scoliosis. In a previous study, we induced bony bridges across the physis of the femur producing an epiphysiodesis in rabbits by using a stimulator modified to deliver a current of 50 μA. The present study builds on this finding to design a model with an aim of inhibiting growth in a unilateral peripheral portion of the vertebral endplate physis, which induces asymmetric spinal growth. Methods-The study was conducted with 8-week-old rabbits; six were treated with electrical current via an implantable 4-lead device; three were age matched normal rabbits. The device was implanted and delivered a constant current of 50 μA from each electrode, continuously for 6 weeks. Weekly radiograph monitoring and endpoint histology were performed. Results-Spinal growth was modified by inducing asymmetric growth of the vertebra of young rabbits using electric stimulators delivering 50 μA of direct current through electrodes implanted in a left peripheral portion of the endplate physis. Conclusion-This concept study, based on our previous study, involved a method and device for inhibiting growth in one aspect of the vertebral endplate using electrical current at an amplitude that induced a hemiepiphysiodesis. Our results demonstrated that this technique both establishes an in vivo model of scoliosis and suggests that if this technique were applied to an existing curve it could potentially induce asymmetrical growth of the spine, thereby correcting scoliosis by continuing the normal growth on the concavity of the curve. Clinical Relevance-A potential new method for modulating spinal growth was developed, and, with further research, this method may be useful in treating children with scoliosis by delivering a growth-inhibiting current to the physeal areas of vertebra through electrodes placed percutaneously.
After cartilage injury and repair, the subchondral bone plate remodels. Skeletal maturity likely ... more After cartilage injury and repair, the subchondral bone plate remodels. Skeletal maturity likely impacts both bone remodeling and inherent cartilage repair capacity. The objective of this study was to evaluate subchondral bone remodeling as a function of injury type, repair scenario, and skeletal maturity in a Yucatan minipig model. Cartilage defects (4 mm) were created bilaterally in the trochlear groove. Treatment conditions included a full thickness chondral defect (full chondral defect, n = 3 adult/3 juvenile), a partial thickness (*50%) chondral defect (PCD, n = 3/3), and FCD treated with microfracture (MFX, n = 3/3). At 6 weeks postoperatively, osteochondral samples containing the lesion site were imaged by micro-computed tomography (CT) and analyzed by histology and immunohistochemistry. Via micro-CT, FCD and MFX groups showed increased bone loss in juveniles compared with adults. Quantification of histology using the ICRS II scoring system showed equal overall assessment for the FCD groups and better overall assessment in juvenile animals treated with MFX compared with adults. All FCD and MFX groups were inferior to control samples. For the PCD injury, both age groups had values close to the control values. For the FCD groups, there were greater alterations in the subchondral bone in juveniles compared with adults. Staining for collagen II showed more intense signals in juvenile FCD and MFX groups compared with adults. This large animal study of cartilage repair shows the significant impact of skeletal maturity on the propensity of subchondral bone to remodel as a result of chondral injury. This will improve selection criteria for animal models for studying cartilage injury, repair, and treatment.
A lack of understanding of the degeneration process has limited treatments aimed at halting or re... more A lack of understanding of the degeneration process has limited treatments aimed at halting or reversing intervertebral disc degeneration. An animal model that replicates key features of human disc degeneration is needed for both elucidating the mechanisms as well as for preclinical testing of therapeutic strategies. The objective of the current work was to investigate the chronic outcomes in an in vivo model in the rat lumbar disc that simulates the diminished glycosaminoglycan and altered mechanics observed in early human disc degeneration. The model had previously been shown to closely resemble early stages of degeneration, yet whether or not the progression to a more severe state occurred was unknown. Rat lumbar intervertebral discs received an injection of a small dose of chondroitinase ABC and evaluated after 24 weeks using microCT to examine disc geometry and abnormal calcifications, disc mechanics, and nucleus glycosaminoglycan content. Additionally, the contribution of aggrecan catabolism was investigated at 4, 12, and 24 weeks through western blots for aggrecan neo-epitope species. Disc height, neutral zone modulus, and range of motion changes that were present at earlier time points were no longer present at 24 weeks. Nucleus glycososaminoglycan remained significantly decreased by 20% relative to controls; however, the magnitude of the decrease was reduced compared to earlier timepoints. The diminished detection of MMP cleaved aggrecan FFGVG fragments after ChABC injection indicates decreased catabolic activity may play a role in the observed restoration of disc properties and function. In summary, an in vivo animal model of early degeneration-like changes was shown to recover towards control levels by 24 weeks. Further work with this model may prove fruitful in defining potential targets for halting or reversing early human degeneration.
Objective. to test different fixation methods of a 3-dimensionally woven poly(ε-caprolactone) (PC... more Objective. to test different fixation methods of a 3-dimensionally woven poly(ε-caprolactone) (PCl) scaffold within chondral defects of a weightbearing large animal model. Methods. Full thickness chondral defects were made in the femoral condyles of 15 adult male Yucatan mini-pigs. two surgical approaches were compared including total arthrotomy (traditional) and a retinaculum-sparing, minimally invasive surgery (MiS) approach. Following microfracture (MFX), scaffolds were placed without fixation or were fixed with fibrin glue, suture, or subchondral anchor. experimental endpoints were between 1 and 6 weeks. Micro-computed tomography and histology were used to assess samples. Results. the MiS approach was superior as the traditional approach caused medial condyle cartilage wear. One of 13 (7.7%) of scaffolds without fixation, 4 of 11 (36.3%) fibrin scaffolds, 1 of 4 (25%) of sutured scaffolds, and 9 of 9 (100%) of anchor-fixed scaffolds remained in place. Histology demonstrated tissue filling with some overgrowth of PCl scaffolds. Conclusions. Of the methods tested, the MiS approach coupled with subchondral anchor fixation provided the best scaffold retention in a mini-pig chondral defect model. this finding has implications for fixation strategies in future animal studies and potential future human use.
Functional repair of focal cartilage defects requires filling the space with neotissue that has c... more Functional repair of focal cartilage defects requires filling the space with neotissue that has compressive properties comparable to native tissue and integration with adjacent host cartilage. While poor integration is a common complication with current clinical treatments, reports of tissue engineering advances in the development of functional compressive properties rarely include analyses of their potential for integration. Our objective was thus to assess both the maturation and integration of mesenchymal stem cell (MSC) laden hyaluronic acid (HA) hydrogels in an in vitro cartilage defect model. Furthermore, we considered the effects of an initial period of prematuration as well as various material formulations to maximize both construct compressive properties and integration strength. MSCs were encapsulated in 1%, 3%, and 5% methacrylated HA (MeHA) or 2% agarose (Ag) and gelled directly (in situ) within an in vitro cartilage defect or were formed and then pre-cultured for 4 weeks before implantation. Results showed the integration strength of pre-cultured repair constructs was equal to (1% MeHA) or greater than (2% Ag) the integration of in situ repaired cartilage. Moreover, MSC chondrogenesis and maturation was restricted by the in situ repair environment with constructs maturing to a much lesser extent than pre-matured constructs. These results indicate that construct pre-maturation may be an essential element of functional cartilage repair.
Cartilage integration remains a clinical challenge for treatment of focal articular defects. Cart... more Cartilage integration remains a clinical challenge for treatment of focal articular defects. Cartilage exhibits limited healing capacity that declines with tissue maturation. Many approaches have been investigated for their ability to stimulate healing of mature cartilage or integration of repair tissue or tissue-engineered constructs with native cartilage. Growth factors present in immature tissue may enhance chondrogenesis and promote integrative repair of cartilage defects. In this study, we assessed the role of one such factor, fibroblast growth factor 18 (FGF18). Studies using FGF18 have shown a variety of positive effects on cartilage, including stimulation of chondrocyte proliferation, matrix biosynthesis, and suppression of proteinase activity. To explore the role of FGF18 on cartilage defect repair, we hypothesized that treatment with recombinant human FGF18 (sprifermin) would increase matrix synthesis in a defect model, thus improving integration strength. To test this hypothesis, 6 mm cartilage cylinders were harvested from juvenile bovine knees. A central 3 mm defect was created in each explant, and this core was removed and replaced. Resulting constructs were cultured in control or sprifermin-containing medium (weekly 24-hour exposure of 100 ng/ml sprifermin) for 4 weeks. Mechanical testing, biochemical analysis, micro-CT, scanning electron microscopy, and histology were used to assess matrix production, adhesive strength, and structural properties of the cartilage-cartilage interface. Results showed greater adhesive strength, increased collagen content, and larger contact areas between core and annular cartilage in the sprifermin-treated group. These findings present a novel treatment for cartilage injuries that have potential to enhance defect healing and lateral cartilage-cartilage integration.
The mechanical properties of the human supraspinatus tendon (SST) are highly heterogeneous and ma... more The mechanical properties of the human supraspinatus tendon (SST) are highly heterogeneous and may reflect an important adaptive response to its complex, multiaxial loading environment. However, these functional properties are associated with a location-dependent structure and composition that have not been fully elucidated. Therefore, the objective of this study was to determine the concentrations of types I, II and III collagen in 6 distinct regions of the SST and compare changes in collagen concentration across regions with local changes in mechanical properties. We hypothesized that type I collagen content would be high throughout the tendon, type II collagen would be restricted to regions of compressive loading and type III collagen content would be high in regions associated with damage. We further hypothesized that regions of high type III collagen content would correspond to regions with low tensile modulus and a low degree of collagen alignment. Although type III collagen content was not significantly higher regions that are frequently damaged, all other hypotheses were supported by our results. In particular, type II collagen content was highest near the insertion while type III collagen was inversely correlated with tendon modulus and collagen alignment. The measured increase in type II collagen under the coracoacromial arch provides evidence of adaptation to compressive loading in the SST. Moreover, the structure-function relationship between type III collagen content and tendon mechanics established in this study demonstrates a mechanism for altered mechanical properties in pathological tendons and provides a guideline for identifying therapeutic targets and pathology-specific biomarkers.
Adult articular cartilage exhibits little capacity for intrinsic repair, and thus even minor inju... more Adult articular cartilage exhibits little capacity for intrinsic repair, and thus even minor injuries or lesions may lead to progressive damage and osteoarthritic joint degeneration, resulting in significant pain and disability. While there have been numerous attempts to develop tissue-engineered grafts or patches to repair focal chondral and osteochondral defects, there remain significant challenges in the clinical application of cell-based therapies for cartilage repair. This paper reviews the current state of cartilage tissue engineering with respect to different cell sources and their potential genetic modification, biomaterial scaffolds and growth factors, as well as preclinical testing in various animal models. This is not intended as a systematic review, rather an opinion of where the field is moving in light of current literature. While significant advances have been made in recent years, the complexity of this problem suggests that a multidisciplinary approach -combining a clinical perspective with expertise in cell biology, biomechanics, biomaterials science and high-throughput analysis will likely be necessary to address the challenge of developing functional cartilage replacements. With this approach we are more likely to realise the clinical goal of treating both focal defects and even large-scale osteoarthritic degenerative changes in the joint.
Cartilage tissue engineering is emerging as a promising treatment for osteoarthritis, and the fie... more Cartilage tissue engineering is emerging as a promising treatment for osteoarthritis, and the field has progressed toward utilizing large animal models for proof of concept and preclinical studies. Mechanical testing of the regenerative tissue is an essential outcome for functional evaluation. However, testing modalities and constitutive frameworks used to evaluate in vitro grown samples differ substantially from those used to evaluate in vivo derived samples. To address this, we developed finite element (FE) models (using FEBio) of unconfined compression and indentation testing, modalities commonly used for such samples. We determined the model sensitivity to tissue radius and subchondral bone modulus, as well as its ability to estimate material parameters using the built-in parameter optimization tool in FEBio. We then sequentially tested agarose gels of 4%, 6%, 8%, and 10% weight/weight using a custom indentation platform, followed by unconfined compression. Similarly, we evaluated the ability of the model to generate material parameters for living constructs by evaluating engineered cartilage. Juvenile bovine mesenchymal stem cells were seeded (2 • 10 7 cells/mL) in 1% weight/volume hyaluronic acid hydrogels and cultured in a chondrogenic medium for 3, 6, and 9 weeks. Samples were planed and tested sequentially in indentation and unconfined compression. The model successfully completed parameter optimization routines for each testing modality for both acellular and cell-based constructs. Traditional outcome measures and the FE-derived outcomes showed significant changes in material properties during the maturation of engineered cartilage tissue, capturing dynamic changes in functional tissue mechanics. These outcomes were significantly correlated with one another, establishing this FE modeling approach as a singular method for the evaluation of functional engineered and native tissue regeneration, both in vitro and in vivo.
Objective: Many approaches are being taken to generate cartilage replacement materials. The goal ... more Objective: Many approaches are being taken to generate cartilage replacement materials. The goal of this study was to use a self-aggregating suspension culture model of chondrocytes with mechanical preconditioning. Design: Our model differs from others in that it is based on a scaffold-less, self-aggregating culture model that produces a cartilage tissue analog that has been shown to share many similarities with the natural cartilage phenotype. Owing to the known loaded environment under which chondrocytes function in vivo, we hypothesized that applying force to the suspension culture-derived chondrocyte biomass would improve its cartilage-like characteristics and provide a new model for engineering cartilage tissue analogs. Results: In this study, we used a specialized hydrostatic pressure bioreactor system to apply mechanical forces during the growth phase to improve biochemical and biophysical properties of the biomaterial formed. We demonstrated that using this high-density suspension culture, a biomaterial more consistent with the hyaline cartilage phenotype was produced without any foreign material added. Unpassaged chondrocytes responded to a physiologically relevant hydrostatic load by significantly increasing gene expression of critical cartilage molecule collagen and aggrecan along with other cartilage relevant genes, CD44, perlecan, decorin, COMP, and iNOS. Conclusions: This study describes a self-aggregating bioreactor model without foreign material or scaffold in which chondrocytes form a cartilage tissue analog with many features similar to native cartilage. This study represents a promising scaffold-less, methodological advancement in cartilage tissue engineering with potential translational applications to cartilage repair.
Background: Post traumatic osteoarthritis is a disabling condition impacting mostly young and act... more Background: Post traumatic osteoarthritis is a disabling condition impacting mostly young and active population. In the present study we investigated the impact of intra-articular sprifermin, a recombinant truncated broblast growth factor 18, on the outcome of microfracture treatment, a widely used surgical technique to enhance cartilage healing at the site of injury. Methods: For the purpose if this study, we created a cartilage defect and performed microfracture treatment in fetlock joints of 18 horses, treated joints with one of three doses of sprifermin (10, 30, or 100 mg) or with saline, hyaluronan (HA), and evaluated animals functional and structural outcomes over 24 weeks. For primary outcome measures we performed histological evaluations and performed gene expression analysis of aggrecan, collagen types I and II, and cartilage oligomeric matrix protein (COMP) in three regions of interest. As secondary outcome measures we examined animal's lameness, performed arthroscopic, radiographic, and CT scan imaging, and gross morphology assessment. Results: We detected the highest treatment bene t following 100 mg sprifermin treatment. Overall histological assessment showed an improvement in the kissing region and expression of constitutive genes showed a concentration dependent enhancement, especially in the peri-lesion area. We detected a signi cant improvement in lameness scores, arthroscopic evaluations, radiography, and CT scans following sprifermin treatment when results from three dose-treatment groups were combined. Conclusion: Our results demonstrated for the rst time, an enhancement in outcomes of the microfracture treatment following sprifermin treatment, suggesting a cartilage regenerative role and a potential bene t of sprifermin treatment in early cartilage injuries.
The repair of focal cartilage defects remains one of the foremost issues in the field of orthopae... more The repair of focal cartilage defects remains one of the foremost issues in the field of orthopaedics. Chondral defects may arise from a variety of joint pathologies and left untreated, will likely progress to osteoarthritis. Current repair techniques, such as microfracture, result in short-term clinical improvements but have poor long-term outcomes. Emerging scaffold-based repair strategies have reported superior outcomes compared to microfracture and motivate the development of new biomaterials for this purpose. In this study, unique composite implants consisting of a base porous reinforcing component (woven poly(ε-caprolactone)) infiltrated with 1 of 2 hydrogels (self-assembling peptide or thermo-gelling hyaluronan) or bone marrow aspirate were evaluated. The objective was to evaluate cartilage repair with composite scaffold treatment compared to the current standard of care (microfracture) in a translationally relevant large animal model, the Yucatan minipig. While many cartilage-repair studies have shown some success in vivo, most are short term and not clinically relevant. Informed by promising 6-week findings, a 12-month study was carried out and those results are presented here. To aid in comparisons across platforms, several structural and functionally relevant outcome measures were performed. Despite positive early findings, the long-term results indicated less than optimal structural and mechanical results with respect to cartilage repair, with all treatment groups performing worse than the standard of care. This study is important in that it brings much needed attention to the importance of performing translationally relevant long-term studies in an appropriate animal model when developing new clinical cartilage repair approaches.
Delivery of biofactors in a precise and controlled fashion remains a clinical challenge. Stimulir... more Delivery of biofactors in a precise and controlled fashion remains a clinical challenge. Stimuliresponsive delivery systems can facilitate 'on-demand' release of therapeutics in response to a variety of physiologic triggering mechanisms (e.g. pH, temperature). However, few systems to date have taken advantage of mechanical inputs from the microenvironment to initiate drug release. Here, we developed mechanically-activated microcapsules (MAMCs) that are designed to deliver therapeutics in an on-demand fashion in response to the mechanically loaded environment of regenerating musculoskeletal tissues, with the ultimate goal of furthering tissue repair. To establish a suite of microcapsules with different thresholds for mechano-activation, we first manipulated MAMC physical dimensions and composition, and evaluated their mechano-response under both direct 2D compression and in 3D matrices mimicking the extracellular matrix properties and dynamic loading environment of regenerating tissue. To demonstrate the feasibility of this delivery system, we used an engineered cartilage model to test the efficacy of mechanically-instigated release of TGF-β3 on the chondrogenesis of mesenchymal stem cells. These data establish a novel platform by which to tune the release of therapeutics and/or
Mucopolysaccharidosis (MPS) VII is a lysosomal storage disorder characterized by deficient βglucu... more Mucopolysaccharidosis (MPS) VII is a lysosomal storage disorder characterized by deficient βglucuronidase activity, which leads to the accumulation of incompletely degraded glycosaminoglycans (GAGs). MPS VII patients present with severe skeletal abnormalities, which are particularly prevalent in the spine. Incomplete cartilage-to-bone conversion in MPS VII vertebrae during postnatal development is associated with progressive spinal deformity and spinal cord compression. The objectives of this study were to determine the earliest postnatal developmental stage at which vertebral bone disease manifests in MPS VII and to identify the underlying cellular basis of impaired cartilage-to-bone conversion, using the naturally-occurring canine model. Control and MPS VII dogs were euthanized at 9 and 14 days-of-age, and vertebral secondary ossification centers analyzed using micro-computed tomography, histology, qPCR, and protein immunoblotting. Imaging studies and mRNA analysis of bone formation markers established that secondary ossification commences between 9 and 14 days in control animals, but not in MPS VII animals. mRNA analysis of differentiation markers revealed that MPS VII epiphyseal *
Background-The concept of modulating spinal growth to correct scoliosis is intriguing, and this s... more Background-The concept of modulating spinal growth to correct scoliosis is intriguing, and this study proposes a new model. Inhibition of vertebral growth on the convex side of a curve would allow continued normal growth on the concave side to correct the scoliosis. In a previous study, we induced bony bridges across the physis of the femur producing an epiphysiodesis in rabbits by using a stimulator modified to deliver a current of 50 μA. The present study builds on this finding to design a model with an aim of inhibiting growth in a unilateral peripheral portion of the vertebral endplate physis, which induces asymmetric spinal growth. Methods-The study was conducted with 8-week-old rabbits; six were treated with electrical current via an implantable 4-lead device; three were age matched normal rabbits. The device was implanted and delivered a constant current of 50 μA from each electrode, continuously for 6 weeks. Weekly radiograph monitoring and endpoint histology were performed. Results-Spinal growth was modified by inducing asymmetric growth of the vertebra of young rabbits using electric stimulators delivering 50 μA of direct current through electrodes implanted in a left peripheral portion of the endplate physis. Conclusion-This concept study, based on our previous study, involved a method and device for inhibiting growth in one aspect of the vertebral endplate using electrical current at an amplitude that induced a hemiepiphysiodesis. Our results demonstrated that this technique both establishes an in vivo model of scoliosis and suggests that if this technique were applied to an existing curve it could potentially induce asymmetrical growth of the spine, thereby correcting scoliosis by continuing the normal growth on the concavity of the curve. Clinical Relevance-A potential new method for modulating spinal growth was developed, and, with further research, this method may be useful in treating children with scoliosis by delivering a growth-inhibiting current to the physeal areas of vertebra through electrodes placed percutaneously.
After cartilage injury and repair, the subchondral bone plate remodels. Skeletal maturity likely ... more After cartilage injury and repair, the subchondral bone plate remodels. Skeletal maturity likely impacts both bone remodeling and inherent cartilage repair capacity. The objective of this study was to evaluate subchondral bone remodeling as a function of injury type, repair scenario, and skeletal maturity in a Yucatan minipig model. Cartilage defects (4 mm) were created bilaterally in the trochlear groove. Treatment conditions included a full thickness chondral defect (full chondral defect, n = 3 adult/3 juvenile), a partial thickness (*50%) chondral defect (PCD, n = 3/3), and FCD treated with microfracture (MFX, n = 3/3). At 6 weeks postoperatively, osteochondral samples containing the lesion site were imaged by micro-computed tomography (CT) and analyzed by histology and immunohistochemistry. Via micro-CT, FCD and MFX groups showed increased bone loss in juveniles compared with adults. Quantification of histology using the ICRS II scoring system showed equal overall assessment for the FCD groups and better overall assessment in juvenile animals treated with MFX compared with adults. All FCD and MFX groups were inferior to control samples. For the PCD injury, both age groups had values close to the control values. For the FCD groups, there were greater alterations in the subchondral bone in juveniles compared with adults. Staining for collagen II showed more intense signals in juvenile FCD and MFX groups compared with adults. This large animal study of cartilage repair shows the significant impact of skeletal maturity on the propensity of subchondral bone to remodel as a result of chondral injury. This will improve selection criteria for animal models for studying cartilage injury, repair, and treatment.
A lack of understanding of the degeneration process has limited treatments aimed at halting or re... more A lack of understanding of the degeneration process has limited treatments aimed at halting or reversing intervertebral disc degeneration. An animal model that replicates key features of human disc degeneration is needed for both elucidating the mechanisms as well as for preclinical testing of therapeutic strategies. The objective of the current work was to investigate the chronic outcomes in an in vivo model in the rat lumbar disc that simulates the diminished glycosaminoglycan and altered mechanics observed in early human disc degeneration. The model had previously been shown to closely resemble early stages of degeneration, yet whether or not the progression to a more severe state occurred was unknown. Rat lumbar intervertebral discs received an injection of a small dose of chondroitinase ABC and evaluated after 24 weeks using microCT to examine disc geometry and abnormal calcifications, disc mechanics, and nucleus glycosaminoglycan content. Additionally, the contribution of aggrecan catabolism was investigated at 4, 12, and 24 weeks through western blots for aggrecan neo-epitope species. Disc height, neutral zone modulus, and range of motion changes that were present at earlier time points were no longer present at 24 weeks. Nucleus glycososaminoglycan remained significantly decreased by 20% relative to controls; however, the magnitude of the decrease was reduced compared to earlier timepoints. The diminished detection of MMP cleaved aggrecan FFGVG fragments after ChABC injection indicates decreased catabolic activity may play a role in the observed restoration of disc properties and function. In summary, an in vivo animal model of early degeneration-like changes was shown to recover towards control levels by 24 weeks. Further work with this model may prove fruitful in defining potential targets for halting or reversing early human degeneration.
Objective. to test different fixation methods of a 3-dimensionally woven poly(ε-caprolactone) (PC... more Objective. to test different fixation methods of a 3-dimensionally woven poly(ε-caprolactone) (PCl) scaffold within chondral defects of a weightbearing large animal model. Methods. Full thickness chondral defects were made in the femoral condyles of 15 adult male Yucatan mini-pigs. two surgical approaches were compared including total arthrotomy (traditional) and a retinaculum-sparing, minimally invasive surgery (MiS) approach. Following microfracture (MFX), scaffolds were placed without fixation or were fixed with fibrin glue, suture, or subchondral anchor. experimental endpoints were between 1 and 6 weeks. Micro-computed tomography and histology were used to assess samples. Results. the MiS approach was superior as the traditional approach caused medial condyle cartilage wear. One of 13 (7.7%) of scaffolds without fixation, 4 of 11 (36.3%) fibrin scaffolds, 1 of 4 (25%) of sutured scaffolds, and 9 of 9 (100%) of anchor-fixed scaffolds remained in place. Histology demonstrated tissue filling with some overgrowth of PCl scaffolds. Conclusions. Of the methods tested, the MiS approach coupled with subchondral anchor fixation provided the best scaffold retention in a mini-pig chondral defect model. this finding has implications for fixation strategies in future animal studies and potential future human use.
Functional repair of focal cartilage defects requires filling the space with neotissue that has c... more Functional repair of focal cartilage defects requires filling the space with neotissue that has compressive properties comparable to native tissue and integration with adjacent host cartilage. While poor integration is a common complication with current clinical treatments, reports of tissue engineering advances in the development of functional compressive properties rarely include analyses of their potential for integration. Our objective was thus to assess both the maturation and integration of mesenchymal stem cell (MSC) laden hyaluronic acid (HA) hydrogels in an in vitro cartilage defect model. Furthermore, we considered the effects of an initial period of prematuration as well as various material formulations to maximize both construct compressive properties and integration strength. MSCs were encapsulated in 1%, 3%, and 5% methacrylated HA (MeHA) or 2% agarose (Ag) and gelled directly (in situ) within an in vitro cartilage defect or were formed and then pre-cultured for 4 weeks before implantation. Results showed the integration strength of pre-cultured repair constructs was equal to (1% MeHA) or greater than (2% Ag) the integration of in situ repaired cartilage. Moreover, MSC chondrogenesis and maturation was restricted by the in situ repair environment with constructs maturing to a much lesser extent than pre-matured constructs. These results indicate that construct pre-maturation may be an essential element of functional cartilage repair.
Cartilage integration remains a clinical challenge for treatment of focal articular defects. Cart... more Cartilage integration remains a clinical challenge for treatment of focal articular defects. Cartilage exhibits limited healing capacity that declines with tissue maturation. Many approaches have been investigated for their ability to stimulate healing of mature cartilage or integration of repair tissue or tissue-engineered constructs with native cartilage. Growth factors present in immature tissue may enhance chondrogenesis and promote integrative repair of cartilage defects. In this study, we assessed the role of one such factor, fibroblast growth factor 18 (FGF18). Studies using FGF18 have shown a variety of positive effects on cartilage, including stimulation of chondrocyte proliferation, matrix biosynthesis, and suppression of proteinase activity. To explore the role of FGF18 on cartilage defect repair, we hypothesized that treatment with recombinant human FGF18 (sprifermin) would increase matrix synthesis in a defect model, thus improving integration strength. To test this hypothesis, 6 mm cartilage cylinders were harvested from juvenile bovine knees. A central 3 mm defect was created in each explant, and this core was removed and replaced. Resulting constructs were cultured in control or sprifermin-containing medium (weekly 24-hour exposure of 100 ng/ml sprifermin) for 4 weeks. Mechanical testing, biochemical analysis, micro-CT, scanning electron microscopy, and histology were used to assess matrix production, adhesive strength, and structural properties of the cartilage-cartilage interface. Results showed greater adhesive strength, increased collagen content, and larger contact areas between core and annular cartilage in the sprifermin-treated group. These findings present a novel treatment for cartilage injuries that have potential to enhance defect healing and lateral cartilage-cartilage integration.
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Papers by George Dodge