Detectable reporter gene expression following transduction of adenovirus and adeno‐associated virus serotype 2 vectors within full‐thickness osteoarthritic and unaffected canine cartilage in vitro and unaffected guinea pig cartilage in vivo

This study quantified and compared the transduction efficiencies of adenoviral (Ad), Arg‐Gly‐Asp (RGD)‐modified Ad, adeno‐associated viral serotype 2 (AAV2), and self‐complementary AAV2 (scAAV2) vectors within full‐thickness osteoarthritic (OA) and unaffected canine cartilage explants in vitro. Intraarticular administration of Ad and scAAV2 vectors was performed to determine the ability of these vectors to transduce unaffected guinea pig cartilage in vivo. Following explant exposure to vector treatment or control, the onset and surface distribution of reporter gene expression was monitored daily with fluorescent microscopy. At termination, explants were divided: one half was digested for analysis using flow cytometry; the remaining portion was used for histology and immunohistochemistry (IHC). Intact articular joints were collected for real‐time RT‐PCR and IHC to detect reporter gene expression following injection of selected vectors. Ad vector transduced focal areas along the perimeters of explants; the remaining vectors transduced chondrocytes across 100% of the surface. Greater mean transduction efficiencies were found with both AAV2 vectors as compared to the Ad vector (p ≤ 0.026). Ad and Ad‐RGD vectors transduced only superficial chondrocytes of OA and unaffected cartilage. Uniform reporter gene expression from AAV2 and scAAV2 was detected in the tangential and transitional zones of OA cartilage, but not deeper zones. AAV2 and scAAV2 vectors achieved partial and full‐thickness transduction of unaffected cartilage. In vivo work revealed that scAAV2 vector, but not Ad vector, transduced deeper zones of cartilage and menisci. This study demonstrates that AAV2 and scAAV2 are reliable vectors for use in cartilage in vitro and in vivo. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:149–155, 2010     

重组腺病毒介导外源性SOX9在正常关节软骨细胞中诱导软骨基质合成的体外表达

背景：关节软骨损害是临床一种常见的损伤,软骨形成转录因子SOX9是一种调控Ⅱ型胶原合成的关键因子。 目的：观察以表达外源性SOX9的腺病毒体外成功感染关节软骨细胞后对Ⅱ型胶原和蛋白聚糖（Aggrecan）合成的影响,探讨软骨损伤后修复软骨缺损的基因治疗方法。 方法：体外构建腺病毒载体AdSOX9和AdGFP,成功感染培养的人关节软骨细胞,分别以逆转录聚合酶链式反应（RT-PCR）技术检测了病毒感染前后SOX9、II型胶原和蛋白聚糖基因mRNA的表达,同时以免疫组化技术检测了病毒感染前后关节软骨细胞中Ⅱ型胶原的表达。 结果：应用AdEasy腺病毒构建专利技术体外成功构建了能高效表达外源性SOX9的腺病毒AdSOX9和只表达绿色荧光蛋白GFP的腺病毒AdGFP;以腺病毒AdSOX9和AdGFP体外成功感染人关节软骨细胞后48h,未感染对照组和AdGFP感染组,均检测到了Ⅱ型胶原和蛋白聚糖的表达;而AdSOX9感染组的细胞中,检测到了SOX9基因mRNA的表达明显增高,与未感染对照组和AdGFP感染组相比有显著性差异（P〈0．05）,同时,Ⅱ型胶原和蛋白聚糖的表达也较未感染对照组和AdGFP感染组明显增高,差异显著（P〈0．05）。 结论：以外源性SOX9为目的基因的腺病毒介导基因治疗方法,在促进关节软骨细胞Ⅱ型胶原和蛋白聚糖合成方面得出了初步满意的结果,SOX9可能是关节软骨缺损基因治疗研究领域一个新的理想靶点,值得继续深入研究。     

Effects of intermittent hydrostatic pressure and BMP‐2 on osteoarthritic human chondrocyte metabolism in vitro

Purpose

This study examined effects of intermittent hydrostatic pressure (IHP) and a chondrogenic growth factor, bone morphogenetic protein‐2 (BMP‐2), on anabolic, catabolic, and other metabolic markers in human osteoarthritic (OA) chondrocytes in vitro.

Methods

Articular chondrocytes, isolated from femoral OA cartilage and maintained in high‐density monolayer culture, were examined for effects of BMP‐2 and IHP on gene expression of matrix‐associated proteins (aggrecan, type II collagen, and SOX9) and catabolic matrix metalloproteinases (MMP‐2 and MMP‐3) and culture medium levels of the metabolic markers MMP‐2, nitric oxide (NO), and glycosaminoglycan (GAG). The results were analyzed using a mixed linear regression model to investigate the effects of load and growth factor concentration.

Results

IHP and BMP‐2 modulated OA chondrocyte metabolism in accordance with growth factor concentration independently, without evidence of synergism or antagonism. Each type of stimulus acted independently on anabolic matrix gene expression. Type II collagen and SOX9 gene expression were stimulated by both IHP and BMP‐2 whereas aggrecan was increased only by BMP‐2. IHP exhibited a trend to decrease MMP‐2 gene expression as a catabolic marker whereas BMP‐2 did not. NO production was increased by addition of BMP‐2 and IHP exhibited a trend for increased levels. GAG production was increased by BMP‐2.

Conclusions

This study confirmed the hypothesis that human OA chondrocytes respond to a specific type of mechanical load, IHP, through enhanced articular cartilage macromolecule gene expression and that IHP, in combination with a chondrogenic growth factor BMP‐2, additively enhanced matrix gene expression without interactive effects. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:361–368, 2011     

Autologous solution protects bovine cartilage explants from IL‐1α‐ and TNFα‐induced cartilage degradation

Osteoarthritis (OA) is characterized by deterioration of articular cartilage driven by an imbalance of pro‐ and anti‐inflammatory cytokines. To address the cartilage deterioration observed in OA, an autologous protein solution (APS) has been developed which has been shown to inhibit the production of destructive proteases and inflammatory cytokines from chondrocytes and monocytes, respectively. The purpose of this study was to determine the chondroprotective effect of APS on IL‐1α‐ or TNFα‐challenged bovine articular cartilage explants. Cartilage explants were cultured in the presence or absence of recombinant inflammatory cytokines, IL‐1α and TNFα. Explants under equivalent inflammatory conditions were pretreated with recombinant antagonists IL‐1ra, sTNF‐RI, or APS to measure their inhibition of matrix degradation. Explants were further evaluated with Safranin‐O, Masson's Trichrome, and Hematoxylin and Eosin histological staining. APS was more effective than recombinant antagonists in preventing cartilage matrix degradation and inhibited any measurable IL‐1α‐induced collagen release over a 21‐day culture period. APS treatment reduced the degree of Safranin‐O staining loss when cartilage explants were cultured with IL‐1α or TNFα. Micrographs of APS treated cartilage explants showed an increase in observed cellularity and apparent cell division. APS may have the potential to prevent cartilage loss associated with early OA. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:1929–1935, 2013     

Intra‐articular resveratrol injection prevents osteoarthritis progression in a mouse model by activating SIRT1 and thereby silencing HIF‐2α

We investigated the feasibility of the intra‐articular injection of resveratrol for preventing the progression of existing cartilage degeneration in a mouse model of osteoarthritis (OA). The effects of resveratrol on the expression of silent information regulator 2 type 1 (SIRT1), hypoxia‐inducible factor‐2α (HIF‐2α) and catabolic factors in OA cartilage was explored. OA was induced in the mouse knee via destabilization of the medial meniscus (DMM). Resveratrol was injected weekly into the operated knee beginning 4 weeks after surgery. The OA phenotype was evaluated via histological and immunohistochemical analyses at 8 weeks after DMM. Western blot analysis was performed to identify whether resveratrol modulated the interleukin (IL)‐1β‐induced expression of HIF‐2α in human chondrocytes. Histologically, resveratrol treatment preserved the structural homeostasis of the articular cartilage and the subchondral bone. Following resveratrol injection, the expression of collagen type II was retained, but the expression of inducible nitric oxide synthase and matrix metalloproteinase‐13 was reduced in OA cartilage. Moreover, the administration of resveratrol significantly induced the activation of SIRT1 and the inhibition of HIF‐2α expression in mouse OA cartilage and in IL‐1β‐treated human chondrocytes. These findings indicate that the intra‐articular injection of resveratrol significantly prevents the destruction of OA cartilage by activating SIRT1 and thereby suppressing the expression of HIF‐2α and catabolic factors. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1061–1070, 2015.     

SOX2基因对骨关节炎软骨细胞凋亡的影响研究

目的 探讨SOX2基因对人骨关节炎（OA）软骨细胞凋亡的影响及机制。方法 以正常软骨组织作为对照,通过Western blotting检测OA软骨组织SOX2蛋白表达。从人OA中分离软骨细胞,参照Lipofectamine TM2000说明将重组体pcDNA3.1-SOX2及空载体pcDNA3.1转染软骨细胞,并设置空白对照组。AG490作为JAK2/STAT3信号通路抑制剂,各组细胞处理48 h,通过流式细胞术、ROS试剂盒分别检测各组细胞凋亡率及ROS水平。Western blotting检测JAK2、p-JAK2、STAT3和p-STAT3的蛋白相对表达量。结果 人OA软骨组织SOX2表达明显低于在正常软骨组织表达（0.065±0.009 vs 0.313±0.028, P<0.05）。转染pcDNA3.1-SOX2的OA软骨细胞SOX2表达明显高于空白组（0.556±0.048 vs 0.122±0.013, P<0.05）。pcDNA3.1-SOX2可明显降低OA软骨细胞凋亡率(3.11±0.42 vs 8.54±0.68)及ROS水平(23.46±2.15 vs 52.67±4.41),上调p-JAK2（0.142±0.013 vs 0.065±0.009）和p-STAT3表达（0.218±0.020 vs 0.126±0.015）（P<0.05）,AG490（15.23±1.13 vs 8.15±0.62）可诱导OA软骨细胞凋亡,而pcDNA3.1-SOX2可减弱AG490对OA软骨细胞凋亡促进作用（P<0.05）。结论 SOX2可抑制OA软骨细胞凋亡,其机制可能与激活JAK2/STAT3信号通路有关。     

TGF-beta-1 gene transfer in joint cartilage cells. Stimulating effect in extracellular matrix synthesis

TGF beta-1 has been shown to upregulate matrix synthesis in articular chondrocytes. TGF beta-gene transfer to chondrocytes has the potential to increase the local production of this key component within regenerating cartilage after trauma and could support the repair process in articular cartilage lesions. Primary rabbit articular chondrocytes were cultured and retrovirally transfected with the experimental TGF beta-1 and the lacZ marker gene for control purposes. After radioactive labeling of new synthesized matrix proteins results were compared with normal primary chondrocytes. After TGF beta-1 gene transfer the endogenous growth factor concentration was doubled compared to normal chondrocytes and decreased in the lacZ control group. The proteoglycan synthesis in TGF beta-1 transfected chondrocytes showed a 96% increase compared to the basal production of normal chondrocytes. The LacZ transfected group revealed the opposite effect by a 44% decrease. The collagen synthesis of TGF beta-1 transfected chondrocytes was 304% compared to normal chondrocytes, predominantly type II collagen. The lacZ group collagen production was reduced by 35%. We conclude that TGF beta-1 gene transfer overcomes the decreasing effect observed by transfection with the LacZ marker gene and increases matrix synthesis in articular chondrocytes. Genetically altered chondrocytes might improve the repair of cartilage lesions by stimulating matrix synthesis and supporting the expression of the hyaline phenotype.     

Biological Properties of Allogenic Articular Chondrocytes on the Surface of Bovine Cartilage Explants in vitro

Bovine cartilage explants were co‐cultured with or without allogenic chondrocytes for 4 weeks. The attachment of the applied chondrocytes to cartilage after labelling with fluorescence was assessed using a confocal laser microscope. Morphological changes and the production of extracellular matrix (ECM) of co‐cultured chondrocytes on intact and damaged surfaces of cartilage were evaluated by histological and immunohistochemical methods. Co‐cultured chondrocytes attached to and proliferated on the intact and damaged areas of cartilage, and a new layer was created there. The defects were also filled with ECM produced by the co‐cultured chondrocytes. Glycosaminoglycans and collagen type II were detected in the newly formed ECM, and large numbers of rounded chondrocytes were observed at primitive lacunae in this matrix at 4 weeks of culture. The results suggest that chondrocytes have the ability to attach to, to proliferate on and to establish a new matrix on the intact and damaged surfaces of cartilage explants.     

Evaluation of adult equine bone marrow‐ and adipose‐derived progenitor cell chondrogenesis in hydrogel cultures

Bone marrow mesenchymal stem cells (BM‐MSCs) and adipose‐derived progenitor cells (ADPCs) are potential alternatives to autologous chondrocytes for cartilage resurfacing strategies. In this study, the chondrogenic potentials of these cell types were compared by quantifying neo‐tissue synthesis and assaying gene expression and accumulation of extracellular matrix (ECM) components of cartilage. Adult equine progenitor cells encapsulated in agarose or self‐assembling peptide hydrogels were cultured in the presence or absence of TGFβ1 for 3 weeks. In BM‐MSCs‐seeded hydrogels, TGFβ1 stimulated ECM synthesis and accumulation 3–41‐fold relative to TGFβ1‐free culture. In ADPC cultures, TGFβ1 stimulated a significant increase in ECM synthesis and accumulation in peptide (18–29‐fold) but not agarose hydrogels. Chromatographic analysis of BM‐MSC‐seeded agarose and peptide hydrogels cultured in TGFβ1 medium showed extensive synthesis of aggrecan‐like proteoglycan monomers. ADPCs seeded in peptide hydrogel also synthesized aggrecan‐like proteoglycans, although to a lesser extent than seen in BM‐MSC hydrogels, whereas aggrecan‐like proteoglycan synthesis in ADPC‐seeded agarose was minimal. RT‐PCR analysis of TGFβ1 cultures showed detectable levels of type II collagen gene expression in BM‐MSC but not ADPC cultures. Histological analysis of TGFβ1‐cultured peptide hydrogels showed the deposition of a continuous proteoglycan‐ and type II collagen rich ECM for BM‐MSCs but not ADPCs. Therefore, this study showed both protein and gene expression evidence of superior chondrogenesis of BM‐MSCs relative to ADPCs. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:322–331, 2008     

Influence of osteoarthritis grade on molecular signature of human cartilage

Articular chondrocytes maintain cartilage matrix turnover and have the capacity for anabolic and catabolic activities that can be influenced by injury and disease. This study tested the hypothesis that catabolic genes are upregulated with regional osteoarthritis (OA) disease severity within a joint. With IRB approval, specimens of knee cartilage obtained as discarded tissues from subjects undergoing arthroplasty were partitioned for each subject by OA disease severity and evaluated for gene expression by RT‐PCR. There was regional OA grade‐associated upregulation of expected inflammatory mediators TNF‐α, TNF receptors, IFN‐γ, and interleukins as well as genes encoding proteolytic enzymes, including Adamts‐5 and MMPs. Osteoclast‐related genes, cathepsin K, tartrate‐resistant acid phosphatase (TRAP), RANKL, RANK, M‐CSF, and c‐fms, but not osteoprotegerin, were induced in advanced grades. In vitro treatment of normal human chondrocytes with interleukin‐1β upregulated similar genes; this provides evidence that chondrocytes per se can be the source of osteoclast‐related factors. Immunohistochemical staining showed that RANK‐ and RANKL‐positive cells were abundant in advanced grades, especially in chondrocyte clusters. This suggests a possible autocrine mechanism by which an osteoclast phenotype is induced in articular chondrocytes. In sum, these studies identified gene expression signatures in human OA cartilage based upon regional disease severity within a joint. There was an effect of OA Grade on expression of osteoclastic lytic enzymes and regulatory factors in human articular chondrocytes. Induction of an osteoclast‐like phenotype in chondrocytes may be part of OA progression and suggests specific therapeutic approaches. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:454–462, 2016.     

Regulation of the friction coefficient of articular cartilage by TGF‐β1 and IL‐1β

Articular cartilage functions to provide a low‐friction surface for joint movement for many decades of life. Superficial zone protein (SZP) is a glycoprotein secreted by chondrocytes in the superficial layer of articular cartilage that contributes to effective boundary lubrication. In both cell and explant cultures, TGF‐β1 and IL‐1β have been demonstrated to, respectively, upregulate and downregulate SZP protein levels. It was hypothesized that the friction coefficient of articular cartilage could also be modulated by these cytokines through SZP regulation. The friction coefficient between cartilage explants (both untreated and treated with TGF‐β1 or IL‐1β) and a smooth glass surface due to sliding in the boundary lubrication regime was measured with a pin‐on‐disk tribometer. SZP was quantified using an enzyme‐linked immunosorbant assay and localized by immunohistochemistry. Both TGF‐β1 and IL‐1β treatments resulted in the decrease of the friction coefficient of articular cartilage in a location‐ and time‐dependent manner. Changes in the friction coefficient due to the TGF‐β1 treatment corresponded to increased depth of SZP staining within the superficial zone, while friction coefficient changes due to the IL‐1β treatment were independent of SZP depth of staining. However, the changes induced by the IL‐1β treatment corresponded to changes in surface roughness, determined from the analysis of surface images obtained with an atomic force microscope. These findings demonstrate that the low friction of articular cartilage can be modified by TGF‐β1 and IL‐1β treatment and that the friction coefficient depends on multiple factors, including SZP localization and surface roughness. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:249–256, 2009     

Tissue neogenesis and STRO‐1 expression in immature and mature articular cartilage

This study determined the potential for neotissue formation and the role of STRO‐1+ cells in immature versus mature articular cartilage. Cartilage explants from immature and mature bovine knee joints were cultured for up to 12 weeks and stained with safranin‐O, for type II collagen and STRO‐1. Bovine chondrocyte pellet cultures and murine knee joints at the age of 2 weeks and 3 months, and surgically injured cartilage, were analyzed for changes in STRO‐1 expression patterns. Results show that immature explants contained more STRO‐1+ cells than mature explants. After 8 weeks in culture, immature explants showed STRO‐1+ cell proliferation and newly formed tissue, which contained glycosaminoglycan and type II collagen. Mature cartilage explants showed only minimal cell expansion and neotissue formation. Pellet cultures with chondrocytes from immature cartilage showed increased glycosaminoglycan synthesis and STRO‐1+ staining, as compared to pellets with mature chondrocytes. The frequency of STRO‐1+ cells in murine knee joints significantly declined with joint maturation. Following surgical injury, immature explants had higher potential for tissue repair than mature explants. In conclusion, these findings suggest that the high percentage of STRO‐1+ cells in immature cartilage changes with joint maturation. STRO‐1+ cells have the potential to form new cartilage spontaneously and after tissue injury. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:96–102, 2010     

Autologous protein solution inhibits MMP‐13 production by IL‐1β and TNFα‐stimulated human articular chondrocytes

Catabolic inflammatory cytokines are prevalent in osteoarthritis (OA). The purpose of this study was to evaluate an autologous protein solution (APS) as a potential chondroprotective agent for OA therapy. APS was prepared from platelet‐rich plasma (PRP). The APS solution contained both anabolic (bFGF, TGF‐β1, TGF‐β2, EGF, IGF‐1, PDGF‐AB, PDGF‐BB, and VEGF) and anti‐inflammatory (IL‐1ra, sTNF‐RI, sTNF‐RII, IL‐4, IL‐10, IL‐13, and IFNγ) cytokines but low concentrations of catabolic cytokines (IL‐1α, IL‐1β, TNFα, IL‐6, IL‐8, IL‐17, and IL‐18). Human articular chondrocytes were pre‐incubated with the antagonists IL‐1ra, sTNF‐RI, or APS prior to the addition of recombinant human IL‐1β or TNFα. Following exposure to inflammatory cytokines, the levels of MMP‐13 in the culture medium were evaluated by ELISA. MMP‐13 production stimulated in chondrocytes by IL‐1β or TNFα was reduced by rhIL‐1ra and sTNF‐RI to near basal levels. APS was also capable of inhibiting the production of MMP‐13 induced by both IL‐1β and TNFα. The combination of anabolic and anti‐inflammatory cytokines in the APS created from PRP may render this formulation to be a potential candidate for the treatment of inflammation in patients at early stages of OA. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1320–1326, 2011     

Abnormal human chondrocyte morphology is related to increased levels of cell‐associated IL‐1β and disruption to pericellular collagen type VI

Early osteoarthritis (OA) is poorly understood, but abnormal chondrocyte morphology might be important. We studied IL‐1β and pericellular collagen type VI in morphologically normal and abnormal chondrocytes. In situ chondrocytes within explants from nondegenerate (grade 0/1) areas of human tibial plateaus (n = 21) were fluorescently labeled and visualized [2‐photon laser scanning microscopy (2PLSM)]. Normal chondrocytes exhibited a “smooth” membrane surface, whereas abnormal cells were defined as demonstrating ≥1 cytoplasmic process. Abnormal chondrocytes were further classified by number and average length of cytoplasmic processes/cell. IL‐1β or collagen type VI associated with single chondrocytes were visualized by fluorescence immuno‐histochemistry and confocal laser scanning microscopy (CLSM). Fluorescence was quantified as the number of positive voxels (i.e., 3D pixels with fluorescence above baseline)/cell. IL‐1β‐associated fluorescence increased between normal and all abnormal cells in the superficial (99.7 ± 29.8 [11 (72)] vs. 784 ± 382 [15 (132)]; p = 0.04, positive voxels/cell) and deep zones (66.5 ± 29.4 [9 (64)] vs. 795 ± 224 [9 (56)]; p = 0.006). There was a correlation (r² = 0.988) between the number of processes/cell (0–5) and IL‐1β, and an increase particularly with short processes (≤5 µm; p = 0.022). Collagen type VI coverage and thickness decreased (p < 0.001 and p = 0.005, respectively) with development of processes. Abnormal chondrocytes in macroscopically nondegenerate cartilage demonstrated a marked increase in IL‐1β and loss of pericellular type VI collagen, changes that could lead to cartilage degeneration. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1507–1514, 2010     

MMP‐mediated collagen breakdown induced by activated protein C in equine cartilage is reduced by corticosteroids

The plasma serine protease activated protein C (APC) is synthesized by human chondrocytes at sites of pathological cartilage fibrillation. APC levels are increased in osteoarthritis (OA) synovial fluid, and in vitro APC has been shown to synergize with interleukin‐1β (IL‐1) to promote degradation from ovine cartilage. A model of equine cartilage degradation was established and used to explore corticosteroid activities. Intraarticular corticosteroids are a commonly prescribed treatment for joint disease, however their role in disease modification remains unclear. APC synergized with IL‐1 or tumor necrosis factor‐α (TNFα), promoting significant collagen degradation from equine cartilage explants within 4 days, but did not augment glycoaminoglycan (GAG) release. APC activated pro‐matrix metalloproteinases (MMP)‐2 but not pro‐MMP‐9, as assessed by gelatin zymography. APC did not directly activate pro‐MMP‐13. Dexamethasone, triamcinolone, and methylprednisolone acetate (MPA) were evaluated at concentrations between 10^{? 5}M and 10^?10M. High concentrations significantly increased GAG release from IL‐1+APC–treated explants. With the exception of MPA at 10^?10M, all concentrations of corticosteroids caused significant decreases in IL‐1+APC‐driven hydroxyproline loss. Treatment with corticosteroids suppressed expression of MMP‐1, ‐3, and ‐13 mRNA. The collagenolysis associated with IL‐1+APC synergy, and the inhibition of this effect by corticosteroids may involve gelatinase activation and downregulation of MMP expression, respectively. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:370–378, 2010     

Mechanical properties and structure–function relationships in articular cartilage repaired using IGF‐I gene‐enhanced chondrocytes

Several studies have demonstrated the benefits of IGF‐I gene therapy in enhancing the histologic and biochemical content of cartilage repaired by chondrocyte transplantation. However, there is little to no data on the mechanical performance of IGF‐I augmented cartilage grafts. This study evaluated the compressive properties of full‐thickness chondral defects in the equine femur repaired with and without IGF‐I gene therapy. Animals were randomly assigned to one of three study cohorts based on chondrocyte treatment provided in each defect: (i) IGF‐I gene delivered by recombinant adeno‐associated virus (rAAV)‐5; (ii) AAV‐5 delivering GFP as a reporter; (iii) naïve cells without virus. In each case, the opposite limb was implanted with a fibrin carrier without cells. Samples were prepared for confined compression testing to measure the aggregate modulus and hydraulic permeability. All treatment groups, regardless of cell content or transduction, had mechanical properties inferior to native cartilage. Overexpression of IGF‐I increased modulus and lowered permeability relative to other treatments. Investigation of structure–property relationships revealed that Ha and k were linearly correlated with GAG content but logarithmically correlated with collagen content. This provides evidence that IGF‐I gene therapy can improve healing of articular cartilage and can greatly increase the mechanical properties of repaired grafts. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:149–153, 2016.     

Novel protein therapeutic joint retention strategy based on collagen‐binding Avimers

Designing drugs to treat diseases associated with articular joints, particularly those targeting chondrocytes, is challenging due to unique local environmental constraints including the avascular nature of cartilage, the absence of a closed joint compartment, and a highly cross‐linked extracellular matrix. In an effort to address these challenges, we developed a novel strategy to prolong residence time of intra‐articularly administered protein therapeutics. Avimer domains are naturally found in membrane polypeptides and mediate diverse protein–protein interactions. Screening of a phage Avimer domain library led to identification of several low affinity type II collagen‐binding Avimers. Following several rounds of mutagenesis and reselection, these initial hits were transformed to high affinity, selective type II collagen‐binding Avimers. One such Avimer (M26) persisted in rat knees for at least 1 month following intra‐articular administration. Fusion of this Avimer to a candidate therapeutic payload, IL‐1Ra, yielded a protein construct which simultaneously bound to type II collagen and to IL‐1 receptor. In vitro, IL‐1Ra_M26 bound selectively to cartilage explants and remained associated even after extensive washing. Binding appeared to occur preferentially to pericellular regions surrounding chondrocytes. An acute intra‐articular IL‐1‐induced IL‐6 challenge rat model was employed to assess in vivo pharmacodynamics. Whereas both IL‐1Ra_M26 and native IL‐1Ra inhibited IL‐6 output when co‐administered with the IL‐1 challenge, only IL‐1Ra_M26 inhibited when administered 1 week prior to IL‐1 challenge. Collagen‐binding Avimers thus represent a promising strategy for enhancing cartilage residence time of protein therapeutics. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1238–1247, 2018.