Harnessing Growth Factor Interactions to Optimize Articular Cartilage Repair.

The failure of cartilage healing is a major impediment to recovery from joint disease or trauma. Growth factors play a central role in cell function and have been proposed as potential therapeutic agents to promote cartilage repair. Decades of investigation have identified many growth factors that promote the formation of cartilage in vitro and in vivo. However, very few of these have progressed to human trials. A growth factor that robustly augments articular cartilage healing remains elusive. This is not surprising. Articular cartilage repair involves multiple cellular processes and it is unlikely that any single agent will be able to optimally regulate all of them. It is more likely that multiple regulatory molecules may be required to optimize the maintenance and restoration of articular cartilage. If this is the case, then interactions among growth factors may be expected to play a key role in determining their therapeutic value. This review explores the hypothesis that growth factor interactions could help optimize articular cartilage healing.

Decreased SIRT1 Activity Is Involved in the Acute Injury Response of Chondrocytes to Ex Vivo Injurious Mechanical Overload.

A better understanding of molecular events following cartilage injury is required to develop treatments that prevent or delay the onset of trauma-induced osteoarthritis. In this study, alterations to SIRT1 activity in bovine articular cartilage explants were evaluated in the 24 h following a mechanical overload, and the effect of pharmacological SIRT1 activator SRT1720 on acute chondrocyte injury was assessed. SIRT1 enzymatic activity decreased as early as 5 min following the mechanical overload, and remained suppressed for at least 24 h. The chondrocyte injury response, including apoptosis, oxidative stress, secretion of inflammatory mediators, and alterations in cartilage matrix expression, was prevented with pharmacological activation of SIRT1 in a dose-dependent manner. Overall, the results implicate SIRT1 deactivation as a key molecular event in chondrocyte injury following a mechanical impact overload. As decreased SIRT1 signaling is associated with advanced age, these findings suggest that downregulated SIRT1 activity may be common to both age-related and injury-induced osteoarthritis.

Hyaluronic acid-binding insulin-like growth factor-1: Creation of a gene encoding a bifunctional fusion protein.

Chondrogenic growth factors are promising therapeutic agents for articular cartilage repair. A persistent impediment to fulfilling this promise is a limited ability to apply and retain the growth factors within the region of cartilage damage that is in need of repair. Current therapies successfully deliver cells and/or matrices, but growth factors are subject to diffusion into the joint space and then loss from the joint. To address this problem, we created a novel gene that encodes a bifunctional fusion protein comprised by a matrix binding domain and a growth factor. The gene encodes the hyaluronic acid binding region of the cartilage matrix molecule, versican, and the chondrogenic growth factor, insulin-like growth factor-1 (IGF-1). We delivered the gene in an adeno-associated virus-based plasmid vector to articular chondrocytes. The cells synthesized and secreted the fusion protein gene product. The fusion protein bound to hyaluronic acid and retained the anabolic and mitogenic actions of IGF-1 on the chondrocytes. This proof-of-concept study suggests that the bifunctional fusion protein, in concert with chondrocytes and a hyaluronic acid-based delivery vehicle, may serve as an intra-articular therapy to help achieve articular cartilage repair.

Regulation of articular chondrocyte catabolic genes by growth factor interaction.

Osteoarthritis is characterized by a loss of articular cartilage homeostasis in which degradation exceeds formation. Several growth factors have been shown to promote cartilage formation by augmenting articular chondrocyte anabolic activity. This study tests the hypothesis that such growth factors also play an anticatabolic role. We transferred individual or combinations of the genes encoding insulin-like growth factor-I, bone morphogenetic protein-2, bone morphogenetic protein-7, transforming growth factor-ß1, and fibroblast growth factor-2, into adult bovine articular chondrocytes and measured the expression of catabolic marker genes encoding A disintegrin and metalloproteinase with thrombospondin motifs-4 and -5, matrix metalloproteinases-3 and -13, and interleukin-6. When delivered individually, or in combination, these growth factor transgenes differentially regulated the direction, magnitude, and time course of expression of the catabolic marker genes. In concert, the growth factor transgenes regulated the marker genes in an interactive fashion that ranged from synergistic inhibition to synergistic stimulation. Synergistic stimulation prevailed over synergistic inhibition, reaching maxima of 15.2- and 2.7-fold, respectively. Neither the magnitude nor the time course of the effect of the transgene combinations could be predicted on the basis of the individual transgene effects. With few exceptions, the data contradict our hypothesis. The results demonstrate that growth factors that are traditionally viewed as chondrogenic tend also to promote catabolic gene expression. The competing actions of these potential therapeutic agents add an additional level of complexity to the selection of regulatory factors for restoring articular cartilage homeostasis or promoting repair.

Human IGF-I propeptide A promotes articular chondrocyte biosynthesis and employs glycosylation-dependent heparin binding.

BACKGROUND: Insulin-like growth factor I (IGF-I) is a key regulator of chondrogenesis, but its therapeutic application to articular cartilage damage is limited by rapid elimination from the repair site. The human IGF-I gene gives rise to three IGF-I propeptides (proIGF-IA, proIGF-IB and proIGF-IC) that are cleaved to create mature IGF-I. In this study, we elucidate the processing of IGF-I precursors by articular chondrocytes, and test the hypotheses that proIGF-I isoforms bind to heparin and regulate articular chondrocyte biosynthesis. METHODS: Human IGF-I propeptides and mutants were overexpressed in bovine articular chondrocytes. IGF-I products were characterized by ELISA, western blot and FPLC using a heparin column. The biosynthetic activity of IGF-I products on articular chondrocytes was assayed for DNA and glycosaminoglycan that the cells produced. RESULTS: Secreted IGF-I propeptides stimulated articular chondrocyte biosynthetic activity to the same degree as mature IGF-I. Of the three IGF-I propeptides, only one, proIGF-IA, strongly bound to heparin. Interestingly, heparin binding of proIGF-IA depended on N-glycosylation at Asn92 in the EA peptide. To our knowledge, this is the first demonstration that N-glycosylation determines the binding of a heparin-binding protein to heparin. CONCLUSION: The biosynthetic and heparin binding abilities of proIGF-IA, coupled with its generation of IGF-I, suggest that proIGF-IA may have therapeutic value for articular cartilage repair. GENERAL SIGNIFICANCE: These data identify human pro-insulin-like growth factor IA as a bifunctional protein. Its combined ability to bind heparin and augment chondrocyte biosynthesis makes it a promising therapeutic agent for cartilage damage due to trauma and osteoarthritis.

Role of sox9 in growth factor regulation of articular chondrocytes.

Chondrogenic polypeptide growth factors influence articular chondrocyte functions that are required for articular cartilage repair. Sox9 is a transcription factor that regulates chondrogenesis, but its role in the growth factor regulation of chondrocyte proliferation and matrix synthesis is poorly understood. We tested the hypotheses that selected chondrogenic growth factors regulate sox9 gene expression and protein production by adult articular chondrocytes and that sox9 modulates the actions of these growth factors. To test these hypotheses, we delivered insulin-like growth factor-I (IGF-I), fibroblast growth factor-2 (FGF-2), bone morphogenetic protein-2 (BMP-2) and/or bone morphogenetic protein-7 (BMP-7), or their respective transgenes to adult bovine articular chondrocytes, and measured changes in sox9 gene expression and protein production. We then knocked down sox9 gene expression with sox9 siRNA, and measured changes in the expression of the genes encoding aggrecan and types I and II collagen, and in the production of glycosaminoglycan, collagen and DNA. We found that FGF-2 or the combination of IGF-I, BMP-2, and BMP-7 increased sox9 gene expression and protein production and that sox9 knockdown modulated growth factor actions in a complex fashion that differed both with growth factors and with chondrocyte function. The data suggest that sox9 mediates the stimulation of matrix production by the combined growth factors and the stimulation of chondrocyte proliferation by FGF-2. The mitogenic effect of the combined growth factors and the catabolic effect of FGF-2 appear to involve sox9-independent mechanisms. Control of these molecular mechanisms may contribute to the treatment of cartilage damage.

Noninvasive assessment of osteoarthritis severity in human explants by multicontrast MRI.

PURPOSE: Medical imaging has the potential to noninvasively diagnose early disease onset and monitor the success of repair therapies. Unfortunately, few reliable imaging biomarkers exist to detect cartilage diseases before advanced degeneration in the tissue. METHOD: In this study, we quantified the ability to detect osteoarthritis (OA) severity in human cartilage explants using a multicontrast magnetic resonance imaging (MRI) approach, inclusive of novel displacements under applied loading by MRI, relaxivity measures, and standard MRI. RESULTS: Displacements under applied loading by MRI measures, which characterized the spatial micromechanical environment by 2D finite and Von Mises strains, were strong predictors of histologically assessed OA severity, both before and after controlling for factors, e.g., patient, joint region, and morphology. Relaxivity measures, sensitive to local macromolecular weight and composition, including T1ρ, but not T1 or T2, were predictors of OA severity. A combined multicontrast approach that exploited spatial variations in tissue biomechanics and extracellular matrix structure yielded the strongest relationships to OA severity. CONCLUSION: Our results indicate that combining multiple MRI-based biomarkers has high potential for the noninvasive measurement of OA severity and the evaluation of potential therapeutic agents used in the treatment of early OA in animal and human trials.

Growth factor transgenes interactively regulate articular chondrocytes.

Adult articular chondrocytes lack an effective repair response to correct damage from injury or osteoarthritis. Polypeptide growth factors that stimulate articular chondrocyte proliferation and cartilage matrix synthesis may augment this response. Gene transfer is a promising approach to delivering such factors. Multiple growth factor genes regulate these cell functions, but multiple growth factor gene transfer remains unexplored. We tested the hypothesis that multiple growth factor gene transfer selectively modulates articular chondrocyte proliferation and matrix synthesis. We tested the hypothesis by delivering combinations of the transgenes encoding insulin-like growth factor I (IGF-I), fibroblast growth factor-2 (FGF-2), transforming growth factor beta1 (TGF-ß1), bone morphogenetic protein-2 (BMP-2), and bone morphogenetic protien-7 (BMP-7) to articular chondrocytes and measured changes in the production of DNA, glycosaminoglycan, and collagen. The transgenes differentially regulated all these chondrocyte activities. In concert, the transgenes interacted to generate widely divergent responses from the cells. These interactions ranged from inhibitory to synergistic. The transgene pair encoding IGF-I and FGF-2 maximized cell proliferation. The three-transgene group encoding IGF-I, BMP-2, and BMP-7 maximized matrix production and also optimized the balance between cell proliferation and matrix production. These data demonstrate an approach to articular chondrocyte regulation that may be tailored to stimulate specific cell functions, and suggest that certain growth factor gene combinations have potential value for cell-based articular cartilage repair.

Growth factor regulation of growth factor production by multiple gene transfer to chondrocytes.

Of the many classes of molecules regulated by growth factors, growth factors themselves are not well investigated. We tested the hypothesis that combinations of endogenous growth factors interactively regulate the production of other growth factors. Growth factors have therapeutic potential for articular cartilage repair, and gene transfer is a promising approach to growth factor delivery. We tested the hypothesis using adult bovine articular chondrocytes treated with combinations of cDNAs encoding insulin-like growth factor I, bone morphogenetic protein-2 and protein-7, transforming growth factor ß1, and fibroblast growth factor 2. We found that these growth factor transgenes regulated each other's growth factor production. This regulation ranged from stimulation to inhibition. Regulation by multiple transgenes was not predictable from the regulatory actions of the individual transgenes. Such interactions may be important for the selection of growth factor genes for cell-based therapies, including articular cartilage repair.

Correlation analysis of cartilage wear with biochemical composition, viscoelastic properties and friction.

Healthy articular cartilage exhibits remarkable resistance to wear, sustaining mechanical loads and relative motion for decades. However, tissues that replace or repair cartilage defects are much less long lasting. Better information on the compositional and material characteristics that contribute to the wear resistance of healthy cartilage could help guide strategies to replace and repair degenerated tissue. The main objective of this study was to assess the relationship between wear of healthy articular cartilage, its biochemical composition, and its viscoelastic material properties. The correlation of these factors with the coefficient of friction during the wear test was also evaluated. Viscoelastic properties of healthy bovine cartilage were determined via stress relaxation indentation. The same specimens underwent an accelerated, in vitro wear test, and the amount of glycosaminoglycans (GAGs) and collagen released during the wear test were considered measures of wear. The frictional response during the wear test was also recorded. The GAG, collagen and water content and the concentration of the enzymatic collagen crosslink pyridinoline were quantified in tissue that was adjacent to each wear test specimen. Finally, correlation analysis was performed to identify potential relationships between wear characteristics of healthy articular cartilage with its composition, viscoelastic material properties and friction. The findings suggest that stiffer cartilage with higher GAG, collagen and water content has a higher wear resistance. Enzymatic collagen crosslinks also enhance the wear resistance of the collagen network. The parameters of wear, composition, and mechanical stiffness of cartilage were all correlated with one another, suggesting that they are interrelated. However, friction was largely independent of these in this study. The results identify characteristics of healthy articular cartilage that contribute to its remarkable wear resistance. These data may be useful for guiding techniques to restore, regenerate, and stabilize cartilage tissue.

Benefits of recombinant adeno-associated virus (rAAV)-mediated insulinlike growth factor I (IGF-I) overexpression for the long-term reconstruction of human osteoarthritic cartilage by modulation of the IGF-I axis.

Administration of therapeutic genes to human osteoarthritic (OA) cartilage is a potential approach to generate effective, durable treatments against this slow, progressive disorder. Here, we tested the ability of recombinant adeno-associated virus (rAAV)-mediated overexpression of human insulinlike growth factor (hIGF)-I to reproduce an original surface in human OA cartilage in light of the pleiotropic activities of the factor. We examined the proliferative, survival and anabolic effects of the rAAV-hIGF-I treatment in primary human normal and OA chondrocytes in vitro and in explant cultures in situ compared with control (reporter) vector delivery. Efficient, prolonged IGF-I secretion via rAAV stimulated the biological activities of OA chondrocytes in all the systems evaluated over extended periods of time, especially in situ, where it allowed for the long-term reconstruction of OA cartilage (at least for 90 d). Remarkably, production of high, stable amounts of IGF-I in OA cartilage using rAAV advantageously modulated the expression of central effectors of the IGF-I axis by downregulating IGF-I inhibitors (IGF binding protein [IGFBP]-3 and IGFBP4) while up-regulating key potentiators (IGFBP5, the IGF-I receptor and downstream mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 [MAPK/ERK-1/2] and phosphatidylinisitol-3/Akt [PI3K/Akt] signal transduction pathways), probably explaining the enhanced responsiveness of OA cartilage to IGF-I treatment. These findings show the benefits of directly providing an IGF-I sequence to articular cartilage via rAAV for the future treatment of human osteoarthritis.

A Photochemical Crosslinking Approach to Enhance Resistance to Mechanical Wear and Biochemical Degradation of Articular Cartilage.

OBJECTIVE: The objective of this study was to evaluate photochemical crosslinking using Al(III) phthalocyanine chloride tetrasulfonic acid (CASPc) and light with a wavelength of 670 nm as a potential therapy to strengthen articular cartilage and prevent tissue degradation. DESIGN: Changes in viscoelastic properties with indentation were used to identify 2 crosslinking protocols for further testing. Crosslinked cartilage was subjected to an in vitro, accelerated wear test. The ability of the crosslinked tissue to resist biochemical degradation via collagenase was also measured. To better understand how photochemical crosslinking with CASPc varies through the depth of the tissue, the distribution of photo-initiator and penetration of light through the tissue depth was characterized. Finally, the effect of CASPc on chondrocyte viability and of co-treatment with an antioxidant was evaluated. RESULTS: The equilibrium modulus was the most sensitive viscoelastic measure of crosslinking. Crosslinking decreased both mechanical wear and collagenase digestion compared with control cartilage. These beneficial effects were realized despite the fact that crosslinking appeared to be localized to a region near the articular surface. In addition, chondrocyte viability was maintained in crosslinked tissue treated with antioxidants. CONCLUSION: These results suggest that photochemical crosslinking with CASPc and 670 nm light holds promise as a potential therapy to prevent cartilage degeneration by protecting cartilage from mechanical wear and biochemical degradation. Limitations were also evident, however, as an antioxidant treatment was necessary to maintain chondrocyte viability in crosslinked tissue.

Inflammatory cytokines and mechanical injury induce post-traumatic osteoarthritis-like changes in a human cartilage-bone-synovium microphysiological system.

BACKGROUND: Traumatic knee injuries in humans trigger an immediate increase in synovial fluid levels of inflammatory cytokines that accompany impact damage to joint tissues. We developed a human in vitro cartilage-bone-synovium (CBS) coculture model to study the role of mechanical injury and inflammation in the initiation of post-traumatic osteoarthritis (PTOA)-like disease. METHODS: Osteochondral plugs (cartilage-bone, CB) along with joint capsule synovium explants (S) were harvested from 25 cadaveric distal femurs from 16 human donors (Collin's grade 0-2, 23-83years). Two-week monocultures (cartilage (C), bone (B), synovium (S)) and cocultures (CB, CBS) were established. A PTOA-like disease group was initiated via coculture of synovium explants with mechanically impacted osteochondral plugs (CBS+INJ, peak stress 5MPa) with non-impacted CB as controls. Disease-like progression was assessed through analyses of changes in cell viability, inflammatory cytokines released to media (10-plex ELISA), tissue matrix degradation, and metabolomics profile. RESULTS: Immediate increases in concentrations of a panel of inflammatory cytokines occurred in CBS+INJ and CBS cocultures and cultures with S alone (IL-1, IL-6, IL-8, and TNF-α among others). CBS+INJ and CBS also showed increased chondrocyte death compared to uninjured CB. The release of sulfated glycosaminoglycans (sGAG) and associated ARGS-aggrecan neoepitope fragments to the medium was significantly increased in CBS and CBS+INJ groups. Distinct metabolomics profiles were observed for C, B, and S monocultures, and metabolites related to inflammatory response in CBS versus CB (e.g., kynurenine, 1-methylnicotinamide, and hypoxanthine) were identified. CONCLUSION: CBS and CBS+INJ models showed distinct cellular, inflammatory, and matrix-related alterations relevant to PTOA-like initiation/progression. The use of human knee tissues from donors that had no prior history of OA disease suggests the relevance of this model in highlighting the role of injury and inflammation in earliest stages of PTOA progression.

Anisotropic properties of articular cartilage in an accelerated in vitro wear test.

Many material properties of articular cartilage are anisotropic, particularly in the superficial zone where collagen fibers have a preferential direction. However, the anisotropy of cartilage wear had not been previously investigated. The objective of this study was to evaluate the anisotropy of cartilage material behavior in an in vitro wear test. The wear and coefficient of friction of bovine condylar cartilage were measured with loading in directions parallel (longitudinal) and orthogonal (transverse) to the collagen fiber orientation at the articular surface. An accelerated cartilage wear test was performed against a T316 stainless-steel plate in a solution of phosphate buffered saline with protease inhibitors. A constant load of 160 N was maintained for 14000 cycles of reciprocal sliding motion at 4 mm/s velocity and a travel distance of 18 mm in each direction. The contact pressure during the wear test was approximately 2 MPa, which is in the range of that reported in the human knee and hip joint. Wear was measured by biochemically quantifying the glycosaminoglycans (GAGs) and collagen that was released from the tissue during the wear test. Collagen damage was evaluated with collagen hybridizing peptide (CHP), while visualization of the tissue composition after the wear test was provided with histologic analysis. Results demonstrated that wear in the transverse direction released about twice as many GAGs than in the longitudinal direction, but that no significant differences were seen in the amount of collagen released from the specimens. Specimens worn in the transverse direction had a higher intensity of CHP stain than those worn in the longitudinal direction, suggesting more collagen damage from wear in the transverse direction. No anisotropy in friction was detected at any point in the wear test. Histologic and CHP images demonstrate that the GAG loss and collagen damage extended through much of the depth of the cartilage tissue, particularly for wear in the transverse direction. These results highlight distinct differences between cartilage wear and the wear of traditional engineering materials, and suggest that further study on cartilage wear is warranted. A potential clinical implication of these results is that orienting osteochondral grafts such that the direction of wear is aligned with the primary fiber direction at the articular surface may optimize the life of the graft.

Production of recombinant AAV vectors encoding insulin-like growth factor I is enhanced by interaction among AAV rep regulatory sequences.

BACKGROUND: Adeno-associated virus (AAV) vectors are promising tools for gene therapy. Currently, their potential is limited by difficulties in producing high vector yields with which to generate transgene protein product. AAV vector production depends in part upon the replication (Rep) proteins required for viral replication. We tested the hypothesis that mutations in the start codon and upstream regulatory elements of Rep78/68 in AAV helper plasmids can regulate recombinant AAV (rAAV) vector production. We further tested whether the resulting rAAV vector preparation augments the production of the potentially therapeutic transgene, insulin-like growth factor I (IGF-I). RESULTS: We constructed a series of AAV helper plasmids containing different Rep78/68 start codon in combination with different gene regulatory sequences. rAAV vectors carrying the human IGF-I gene were prepared with these vectors and the vector preparations used to transduce HT1080 target cells. We found that the substitution of ATG by ACG in the Rep78/68 start codon in an AAV helper plasmid (pAAV-RC) eliminated Rep78/68 translation, rAAV and IGF-I production. Replacement of the heterologous sequence upstream of Rep78/68 in pAAV-RC with the AAV2 endogenous p5 promoter restored translational activity to the ACG mutant, and restored rAAV and IGF-I production. Insertion of the AAV2 p19 promoter sequence into pAAV-RC in front of the heterologous sequence also enabled ACG to function as a start codon for Rep78/68 translation. The data further indicate that the function of the AAV helper construct (pAAV-RC), that is in current widespread use for rAAV production, may be improved by replacement of its AAV2 unrelated heterologous sequence with the native AAV2 p5 promoter. CONCLUSION: Taken together, the data demonstrate an interplay between the start codon and upstream regulatory sequences in the regulation of Rep78/68 and indicate that selective mutations in Rep78/68 regulatory elements may serve to augment the therapeutic value of rAAV vectors.

Comparative Effectiveness of Structural versus Regulatory Protein Gene Transfer on Articular Chondrocyte Matrix Gene Expression.

OBJECTIVE: The production of extracellular matrix is a necessary component of articular cartilage repair. Gene transfer is a promising method to improve matrix biosynthesis by articular chondrocytes. Gene transfer may employ transgenes encoding regulatory factors that stimulate the production of matrix proteins, or may employ transgenes that encode the proteins themselves. The objective of this study was to determine which of these 2 approaches would be the better choice for further development. We compared these 2 approaches using the transgenes encoding the structural matrix proteins, aggrecan or type II collagen, and the transgene encoding the anabolic factor, insulin-like growth factor I (IGF-I). METHODS: We transfected adult bovine articular chondrocytes with constructs encoding type II collagen, aggrecan, or IGF-I, and measured the expression of type II collagen ( COL2A1) and aggrecan ( ACAN) from their native genes and from their transgenes. RESULTS: IGF-I gene ( IGF1) transfer increased the expression of the native chondrocyte COL2A1 and ACAN genes 2.4 and 2.9 times control, respectively. COL2A1 gene transfer did not significantly increase COL2A1 transcripts, even when the transgene included the genomic COL2A1 regulatory sequences stimulated by chondrogenic growth factors. In contrast, ACAN gene transfer increased ACAN transcripts up to 3.4 times control levels. IGF1, but not ACAN, gene transfer increased aggrecan protein production. CONCLUSION: Taken together, these results suggest that the type II collagen and aggrecan production required for articular cartilage repair will be more effectively achieved by genes that encode anabolic regulatory factors than by genes that encode the matrix molecules themselves.

Bone morphogenetic protein-2 and -9 regulate the interaction of insulin-like growth factor-I with growth plate chondrocytes.

Insulin-like growth factor-I (IGF-I) is thought to play an important role in skeletal growth and development through its mitogenic and anabolic effects on epiphyseal growth plate chondrocytes. The bone morphogenetic proteins (BMPs) have been shown to promote endochondral osteogenesis, and some members of the BMP family, including BMP-2 and BMP-9, have anabolic effects on chondrocyte metabolism. We tested the hypothesis that BMP-2 and BMP-9 interact with IGF-I to modulate growth plate chondrocyte mitotic activity. IGF-I, but neither BMP-2 nor BMP-9, stimulated chondrocyte DNA synthesis. However, both BMP-2 and BMP-9 augmented the mitogenic action of IGF-I. BMP-2, but not BMP-9 increased IGF-I binding to growth plate chondrocytes in kinetic studies. In affinity labeling studies, 125I-IGF-I predominantly labeled an Mr approximately 135-kDa moiety, consistent with the alpha subunit of the type 1 IGF receptor and an Mr approximately 250-kDa moiety consistent with the type 2 IGF receptor. 125I-IGF-I labeling also appeared at Mr approximately 43 kDa, consistent with 125I-IGF-I binding to insulin-like growth binding protein-3. Treatment of chondrocytes with BMP-2, but not with BMP-9, increased the intensity of the Mr approximately 135-kDa band and decreased the intensity of the Mr approximately 43-kDa band. Taken together, these data suggest that the BMPs may modulate the action of IGF-I via the type 1 IGF receptor and/or IGF binding proteins.

Customized biomaterials to augment chondrocyte gene therapy.

A persistent challenge in enhancing gene therapy is the transient availability of the target gene product. This is particularly true in tissue engineering applications. The transient exposure of cells to the product could be insufficient to promote tissue regeneration. Here we report the development of a new material engineered to have a high affinity for a therapeutic gene product. We focus on insulin-like growth factor-I (IGF-I) for its highly anabolic effects on many tissues such as spinal cord, heart, brain and cartilage. One of the ways that tissues store IGF-I is through a group of insulin like growth factor binding proteins (IGFBPs), such as IGFBP-5. We grafted the IGF-I binding peptide sequence from IGFBP-5 onto alginate in order to retain the endogenous IGF-I produced by transfected chondrocytes. This novel material bound IGF-I and released the growth factor for at least 30days in culture. We found that this binding enhanced the biosynthesis of transfected cells up to 19-fold. These data demonstrate the coordinated engineering of cell behavior and material chemistry to greatly enhance extracellular matrix synthesis and tissue assembly, and can serve as a template for the enhanced performance of other therapeutic proteins. STATEMENT OF SIGNIFICANCE: The present manuscript focuses on the enhancement of chondrocyte gene therapy through the modification of scaffold materials to enhance the retention of targeted gene products. This study combined tissue engineering and gene therapy, where customized biomaterials augmented the action of IGF-I by enhancing the retention of protein produced by transfection of the IGF-I gene. This approach enabled tuning of binding of IGF-I to alginate, which increased GAG and HYPRO production by transfected chondrocytes. To our knowledge, peptide-based modification of materials to augment growth factor-targeted gene therapy has not been reported previously.

Genipin crosslinking decreases the mechanical wear and biochemical degradation of impacted cartilage in vitro.

High energy trauma to cartilage causes surface fissures and microstructural damage, but the degree to which this damage renders the tissue more susceptible to wear and contributes to the progression of post-traumatic osteoarthritis (PTOA) is unknown. Additionally, no treatments are currently available to strengthen cartilage after joint trauma and to protect the tissue from subsequent degradation and wear. The purposes of this study were to investigate the role of mechanical damage in the degradation and wear of cartilage, to evaluate the effects of impact and subsequent genipin crosslinking on the changes in the viscoelastic parameters of articular cartilage, and to test the hypothesis that genipin crosslinking is an effective treatment to enhance the resistance to biochemical degradation and mechanical wear. Results demonstrate that cartilage stiffness decreases after impact loading, likely due to the formation of fissures and microarchitectural damage, and is partially or fully restored by crosslinking. The wear resistance of impacted articular cartilage was diminished compared to undamaged cartilage, suggesting that mechanical damage that is directly induced by the impact may contribute to the progression of PTOA. However, the decrease in wear resistance was completely reversed by the crosslinking treatments. Additionally, the crosslinking treatments improved the resistance to collagenase digestion at the impact-damaged articular surface. These results highlight the potential therapeutic value of collagen crosslinking via genipin in the prevention of cartilage degeneration after traumatic injury. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:558-565, 2017.

In vivo articular cartilage deformation: noninvasive quantification of intratissue strain during joint contact in the human knee.

The in vivo measurement of articular cartilage deformation is essential to understand how mechanical forces distribute throughout the healthy tissue and change over time in the pathologic joint. Displacements or strain may serve as a functional imaging biomarker for healthy, diseased, and repaired tissues, but unfortunately intratissue cartilage deformation in vivo is largely unknown. Here, we directly quantified for the first time deformation patterns through the thickness of tibiofemoral articular cartilage in healthy human volunteers. Magnetic resonance imaging acquisitions were synchronized with physiologically relevant compressive loading and used to visualize and measure regional displacement and strain of tibiofemoral articular cartilage in a sagittal plane. We found that compression (of 1/2 body weight) applied at the foot produced a sliding, rigid-body displacement at the tibiofemoral cartilage interface, that loading generated subject- and gender-specific and regionally complex patterns of intratissue strains, and that dominant cartilage strains (approaching 12%) were in shear. Maximum principle and shear strain measures in the tibia were correlated with body mass index. Our MRI-based approach may accelerate the development of regenerative therapies for diseased or damaged cartilage, which is currently limited by the lack of reliable in vivo methods for noninvasive assessment of functional changes following treatment.