Ephrin B2/EphB4 Mediates the Actions of IGF‐I Signaling in Regulating Endochondral Bone Formation

Ephrin B2/EphB4 mediates interactions among osteoblasts (OBs), osteoclasts (OCLs), and chondrocytes to regulate their differentiation. We investigated the role of ephrin B2/EphB4 signaling in mediating the anabolic effects of insulin‐like growth factor‐I (IGF‐I) and parathyroid hormone (PTH) on those cells and overall endochondral bone formation. Immunohistochemistry demonstrated that the expression of ephrin B2 in OBs, OCLs, and osteocytes, and the expression of EphB4 in OBs and osteocytes was dramatically decreased in global IGF‐I knockout mice. Inactivation of EphB4 by EphB4 small, interfering RNA (siRNA) in cultured bone marrow stromal cells significantly decreased the mRNA levels of OB differentiation markers and abolished the stimulatory effects of IGF‐I on these markers. Blocking the interaction of EphB4 and ephrin B2 in the OB‐OCL cocultures with the EphB4 specific peptide TNYL‐RAW or deletion of ephrin B2 in OCL prior to coculture led to fewer and smaller tartrate‐resistant acid phosphatase (TRAP)‐positive cells, decreased expression of OB differentiation markers, and blunted response to IGF‐I for both OCL and OB differentiation. In the growth plate, both ephrin B2 and EphB4 are expressed in late stage proliferating and prehypertrophic chondrocytes, and their expression was decreased in mice lacking the IGF‐I receptor specifically in chondrocytes. In vitro, blocking the interaction of EphB4 and ephrin B2 in chondrogenic ATDC5 cells with TNYL‐RAW significantly decreased both basal and IGF1‐induced expression of type II and type X collagen. In the cocultures of ATDC5 cells and spleen cells (osteoclast precursors), TNYL‐RAW decreased the numbers of TRAP‐positive cells and the expression of nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) and receptor activator of NF‐κB (RANK), and blocked their stimulation by IGF‐I. Our data indicate that IGF‐I/IGF‐IR signaling promotes OB, OCL, and chondrocyte differentiation via ephrin B2/EphB4 mediated cell‐cell communication. © 2014 American Society for Bone and Mineral Research.     

IGF‐1 Regulates Vertebral Bone Aging Through Sex‐Specific and Time‐Dependent Mechanisms

Advanced aging is associated with increased risk of bone fracture, especially within the vertebrae, which exhibit significant reductions in trabecular bone structure. Aging is also associated with a reduction in circulating levels of insulin‐like growth factor (IGF‐1). Studies have suggested that the reduction in IGF‐1 compromises healthspan, whereas others report that loss of IGF‐1 is beneficial because it increases healthspan and lifespan. To date, the effect of decreases in circulating IGF‐1 on vertebral bone aging has not been thoroughly investigated. Here, we delineate the consequences of a loss of circulating IGF‐1 on vertebral bone aging in male and female Igf^f/f mice. IGF‐1 was reduced at multiple specific time points during the mouse lifespan: early in postnatal development (crossing albumin–cyclic recombinase [Cre] mice with Igf^f/f mice); and in early adulthood and in late adulthood using hepatic‐specific viral vectors (AAV8‐TBG‐Cre). Vertebrae bone structure was analyzed at 27 months of age using micro–computed tomography (μCT) and quantitative bone histomorphometry. Consistent with previous studies, both male and female mice exhibited age‐related reductions in vertebral bone structure. In male mice, reduction of circulating IGF‐1 induced at any age did not diminish vertebral bone loss. Interestingly, early‐life loss of IGF‐1 in females resulted in a 67% increase in vertebral bone volume fraction, as well as increased connectivity density and increased trabecular number. The maintenance of bone structure in the early‐life IGF‐1–deficient females was associated with increased osteoblast surface and an increased ratio of osteoprotegerin/receptor‐activator of NF‐κB‐ligand (RANKL) levels in circulation. Within 3 months of a loss of IGF‐1, there was a 2.2‐fold increase in insulin receptor expression within the vertebral bones of our female mice, suggesting that local signaling may compensate for the loss of circulating IGF‐1. Together, these data suggest the age‐related loss of vertebral bone density in females can be reduced by modifying circulating IGF‐1 levels early in life. © 2015 American Society for Bone and Mineral Research.     

Elevated serum IGF‐1 levels synergize PTH action on the skeleton only when the tissue IGF‐1 axis is intact

There is growing evidence that insulin‐like growth factor 1 (IGF‐1) and parathyroid hormone (PTH) have synergistic actions on bone and that part of the anabolic effects of PTH is mediated by local production of IGF‐1. In this study we analyzed the skeletal response to PTH in mouse models with manipulated endocrine or autocrine/paracrine IGF‐1. We used mice carrying a hepatic IGF‐1 transgene (HIT), which results in a threefold increase in serum IGF‐1 levels and normal tissue IGF‐1 expression, and Igf1 null mice with blunted IGF‐1 expression in tissues but threefold increases in serum IGF‐1 levels (KO‐HIT). Evaluation of skeletal growth showed that elevations in serum IGF‐1 in mice with Igf1 gene ablation in all tissues except the liver (KO‐HIT) resulted in a restoration of skeletal morphology and mechanical properties by adulthood. Intermittent PTH treatment of adult HIT mice resulted in increases in serum osteocalcin levels, femoral total cross‐sectional area, cortical bone area and cortical bone thickness, as well as bone mechanical properties. We found that the skeletal response of HIT mice to PTH was significantly higher than that of control mice, suggesting synergy between IGF‐1 and PTH on bone. In sharp contrast, although PTH‐treated KO‐HIT mice demonstrated an anabolic response in cortical and trabecular bone compartments compared with vehicle‐treated KO‐HIT mice, their response was identical to that of PTH‐treated control mice. We conclude that (1) in the presence of elevated serum IGF‐1 levels, PTH can exert an anabolic response in bone even in the total absence of tissue IGF‐1, and (2) elevations in serum IGF‐1 levels synergize PTH action on bone only if the tissue IGF‐1 axis is intact. Thus enhancement of PTH anabolic actions depends on tissue IGF‐1. © 2010 American Society for Bone and Mineral Research.     

Mesenchymal stem cells and insulin‐like growth factor‐I gene‐enhanced mesenchymal stem cells improve structural aspects of healing in equine flexor digitorum superficialis tendons

Tendinitis remains a catastrophic injury among athletes. Mesenchymal stem cells (MSCs) have recently been investigated for use in the treatment of tendinitis. Previous work has demonstrated the value of insulin‐like growth factor‐I (IGF‐I) to stimulate cellular proliferation and tendon fiber deposition in the core lesion of tendinitis. This study examined the effects of MSCs, as well as IGF‐I gene‐enhanced MSCs (AdIGF‐MSCs) on tendon healing in vivo. Collagenase‐induced bilateral tendinitis lesions were created in equine flexor digitorum superficialis tendons (SDFT). Tendons were treated with 10 × 10⁶ MSCs or 10 × 10⁶ AdIGF‐MSCs. Control limbs were injected with 1 mL of phosphate‐buffered saline (PBS). Ultrasound examinations were performed at t = 0, 2, 4, 6, and 8 weeks. Horses were euthanized at 8 weeks and SDFTs were mechanically tested to failure and evaluated for biochemical composition and histologic characteristics. Expression of collagen types I and III, IGF‐I, cartilage oligomeric matrix protein (COMP), matrix metalloproteinase‐3 (MMP‐3), matrix metalloproteinase‐13 (MMP‐13), and aggrecanase‐1 (ADAMTS‐4) were similar in MSC and control tendons. Both MSC and AdIGF‐MSC injection resulted in significantly improved tendon histological scores. These findings indicate a benefit to the use of MSCs and AdIGF‐MSCs for the treatment of tendinitis. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:1392–1398, 2009     

Cell line IDG‐SW3 replicates osteoblast‐to‐late‐osteocyte differentiation in vitro and accelerates bone formation in vivo

Osteocytes are the most abundant cells in bone yet are the most challenging to study because they are embedded in a mineralized matrix. We generated a clonal cell line called IDG‐SW3 (for Immortomouse/Dmp1‐GFP‐SW3) from long‐bone chips from mice carrying a Dmp1 promoter driving GFP crossed with the Immortomouse, which expresses a thermolabile SV40 large T antigen regulated by interferon γ (IFN‐γ). Cells from these mice can be expanded at 33 °C in the presence of IFN‐γ and then allowed to resume their original phenotype at 37 °C in the absence of IFN‐γ. IDG‐SW3 cells are Dmp1‐GFP^? and T antigen⁺ under immortalizing conditions but Dmp1‐GFP⁺ and T antigen^? under osteogenic conditions. Like osteoblasts, they express alkaline phosphatase and produce and mineralize a type 1 collagen matrix containing calcospherulites. Like early osteocytes, they express E11/gp38, Dmp1, MEPE, and Phex. Like late osteocytes, they develop a dendritic morphology and express SOST/sclerostin and fibroblast growth factor 23 (FGF‐23), regulated by parathyroid hormone (PTH) and 1,25‐dihydroxyvitamin D₃. When cultured on 3D matrices, they express Dmp1‐GFP and sclerostin. When the 3D cultures are implanted in calvarial defects in vivo, they accelerate bone healing. This cell line should prove useful for studying osteoblast‐to‐osteocyte transition, mechanisms for biomineralization, osteocyte function, and regulation of SOST/sclerostin and FGF‐23. © 2011 American Society for Bone and Mineral Research     

Gender‐Specific Differences in the Skeletal Response to Continuous PTH in Mice Lacking the IGF1 Receptor in Mature Osteoblasts

The primary goal of this study was to determine whether the IGF1R in mature osteoblasts and osteocytes was required for the catabolic actions of continuous parathyroid hormone (cPTH). Igf1r was deleted from male and female FVN/B mice by breeding with mice expressing cre recombinase under control of the osteocalcin promoter (^0CNIgfr1^‐/‐). Littermates lacking the cre recombinase served as controls. PTH, 60 μg/kg/d, was administered continuously by Alzet minipumps for 4 weeks. Blood was obtained for indices of calcium metabolism. The femurs were examined by micro‐computed tomography for structure, immunohistochemistry for IGF1R expression, histomorphometry for bone formation rates (BFR), mRNA levels by qPCR, and bone marrow stromal cell cultures (BMSC) for alkaline phosphatase activity (ALP⁺), mineralization, and osteoblast‐induced osteoclastogenesis. Whereas cPTH led to a reduction in trabecular bone volume/tissue volume (BV/TV) and cortical thickness in the control females, no change was found in the control males. Although trabecular BV/TV and cortical thickness were reduced in the ^0CNIgfr1^‐/‐ mice of both sexes, no further reduction after cPTH was found in the females, unlike the reduction in males. BFR was stimulated by cPTH in the controls but blocked by Igf1r deletion in the females. The ^0CNIgfr1^‐/‐ male mice showed a partial response. ALP⁺ and mineralized colony formation were higher in BMSC from control males than from control females. These markers were increased by cPTH in both sexes, but BMSC from male ^0CNIgfr1^‐/‐ also were increased by cPTH, unlike those from female ^0CNIgfr1^‐/‐. cPTH stimulated receptor activator of NF‐κB ligand (RANKL) and decreased osteoprotegerin and alkaline phosphatase expression more in control female bone than in control male bone. Deletion of Igf1r blocked these effects of cPTH in the female but not in the male. However, PTH stimulation of osteoblast‐driven osteoclastogenesis was blocked by deleting Igfr1 in both sexes. We conclude that cPTH is catabolic in female but not male mice. Moreover, IGF1 signaling plays a greater role in the skeletal actions of cPTH in the female mouse than in the male mouse, which may underlie the sex differences in the response to cPTH. © 2015 American Society for Bone and Mineral Research.     

The role of estrogen receptor α in growth plate cartilage for longitudinal bone growth

Estrogens enhance skeletal growth during early sexual maturation, whereas high estradiol levels during late puberty result in growth plate fusion in humans. Although the growth plates do not fuse directly after sexual maturation in rodents, a reduction in growth plate height is seen by treatment with a high dose of estradiol. It is unknown whether the effects of estrogens on skeletal growth are mediated directly via estrogen receptors (ERs) in growth plate cartilage and/or indirectly via other mechanisms such as the growth hormone/insulin‐like growth factor 1 (GH/IGF‐1) axis. To determine the role of ERα in growth plate cartilage for skeletal growth, we developed a mouse model with cartilage‐specific inactivation of ERα. Although mice with total ERα inactivation displayed affected longitudinal bone growth associated with alterations in the GH/IGF‐1 axis, the skeletal growth was normal during sexual maturation in mice with cartilage‐specific ERα inactivation. High‐dose estradiol treatment of adult mice reduced the growth plate height as a consequence of attenuated proliferation of growth plate chondrocytes in control mice but not in cartilage‐specific ERα^?/? mice. Adult cartilage‐specific ERα^?/? mice continued to grow after 4 months of age, whereas growth was limited in control mice, resulting in increased femur length in 1‐year‐old cartilage‐specific ERα^?/? mice compared with control mice. We conclude that during early sexual maturation, ERα in growth plate cartilage is not important for skeletal growth. In contrast, it is essential for high‐dose estradiol to reduce the growth plate height in adult mice and for reduction of longitudinal bone growth in elderly mice. © 2010 American Society for Bone and Mineral Research.     

Silent information regulator (Sir)T1 inhibits NF‐κB signaling to maintain normal skeletal remodeling

Silent information regulator T1 (SirT1) is linked to longevity and negatively controls NF‐κB signaling, a crucial mediator of survival and regulator of both osteoclasts and osteoblasts. Here we show that NF‐κB repression by SirT1 in both osteoclasts and osteoblasts is necessary for proper bone remodeling and may contribute to the mechanisms linking aging and bone loss. Osteoclast‐ or osteoblast‐specific SirT1 deletion using the Sirt^flox/flox mice crossed to lysozyme M‐cre and the 2.3 kb col1a1‐cre transgenic mice, respectively, resulted in decreased bone mass caused by increased resorption and reduced bone formation. In osteoclasts, lack of SirT1 promoted osteoclastogenesis in vitro and activated NF‐κB by increasing acetylation of Lysine 310. Importantly, this increase in osteoclastogenesis was blocked by pharmacological inhibition of NF‐κB. In osteoblasts, decreased SirT1 reduced osteoblast differentiation, which could also be rescued by inhibition of NF‐κB. In further support of the critical role of NF‐κB signaling in bone remodeling, elevated NF‐κB activity in IκBα^+/? mice uncoupled bone resorption and formation, leading to reduced bone mass. These findings support the notion that SirT1 is a genetic determinant of bone mass, acting in a cell‐autonomous manner in both osteoblasts and osteoclasts, through control of NF‐κB and bone cell differentiation. © 2013 American Society for Bone and Mineral Research.     

Intermittent PTH Administration Stimulates Pre‐Osteoblastic Proliferation Without Leading to Enhanced Bone Formation in Osteoclast‐Less c‐fos−/− Mice

This study aimed to investigate the behavior and ultrastructure of osteoblastic cells after intermittent PTH treatment and attempted to elucidate the role of osteoclasts on the mediation of PTH‐driven bone anabolism. After administering PTH intermittently to wildtype and c‐fos^?/? mice, immunohistochemical, histomorphometrical, ultrastructural, and statistical examinations were performed. Structural and kinetic parameters related to bone formation were increased in PTH‐treated wildtype mice, whereas in the osteoclast‐deficient c‐fos^?/? mice, there were no significant differences between groups. In wildtype and knockout mice, PTH administration led to significant increases in the number of cells double‐positive for alkaline phosphatase and BrdU, suggesting active pre‐osteoblastic proliferation. Ultrastructural examinations showed two major pre‐osteoblastic subtypes: one rich in endoplasmic reticulum (ER), the hypER cell, and other with fewer and dispersed ER, the misER cell. The latter constituted the most abundant preosteoblastic phenotype after PTH administration in the wildtype mice. In c‐fos^?/? mice, misER cells were present on the bone surfaces but did not seem to be actively producing bone matrix. Several misER cells were shown to be positive for EphB4 and were eventually seen rather close to osteoclasts in the PTH‐administered wildtype mice. We concluded that the absence of osteoclasts in c‐fos^?/? mice might hinder PTH‐driven bone anabolism and that osteoclastic presence may be necessary for full osteoblastic differentiation and enhanced bone formation seen after intermittent PTH administration.     

Activation of β‐Catenin Signaling in Articular Chondrocytes Leads to Osteoarthritis‐Like Phenotype in Adult β‐Catenin Conditional Activation Mice

Osteoarthritis (OA) is a degenerative joint disease, and the mechanism of its pathogenesis is poorly understood. Recent human genetic association studies showed that mutations in the Frzb gene predispose patients to OA, suggesting that the Wnt/β‐catenin signaling may be the key pathway to the development of OA. However, direct genetic evidence for β‐catenin in this disease has not been reported. Because tissue‐specific activation of the β‐catenin gene (targeted by Col2a1‐Cre) is embryonic lethal, we specifically activated the β‐catenin gene in articular chondrocytes in adult mice by generating β‐catenin conditional activation (cAct) mice through breeding of β‐catenin^{fx(Ex3)/fx(Ex3)} mice with Col2a1‐CreER^T2 transgenic mice. Deletion of exon 3 of the β‐catenin gene results in the production of a stabilized fusion β‐catenin protein that is resistant to phosphorylation by GSK‐3β. In this study, tamoxifen was administered to the 3‐ and 6‐mo‐old Col2a1‐CreER^T2;β‐catenin^fx(Ex3)/wt mice, and tissues were harvested for histologic analysis 2 mo after tamoxifen induction. Overexpression of β‐catenin protein was detected by immunostaining in articular cartilage tissues of β‐catenin cAct mice. In 5‐mo‐old β‐catenin cAct mice, reduction of Safranin O and Alcian blue staining in articular cartilage tissue and reduced articular cartilage area were observed. In 8‐mo‐old β‐catenin cAct mice, cell cloning, surface fibrillation, vertical clefting, and chondrophyte/osteophyte formation were observed. Complete loss of articular cartilage layers and the formation of new woven bone in the subchondral bone area were also found in β‐catenin cAct mice. Expression of chondrocyte marker genes, such as aggrecan, Mmp‐9, Mmp‐13, Alp, Oc, and colX, was significantly increased (3‐ to 6‐fold) in articular chondrocytes derived from β‐catenin cAct mice. Bmp2 but not Bmp4 expression was also significantly upregulated (6‐fold increase) in these cells. In addition, we also observed overexpression of β‐catenin protein in the knee joint samples from patients with OA. These findings indicate that activation of β‐catenin signaling in articular chondrocytes in adult mice leads to the premature chondrocyte differentiation and the development of an OA‐like phenotype. This study provides direct and definitive evidence about the role of β‐catenin in the development of OA.     

The quantitative and functional relation between insulin‐like growth factor‐I (IGF) and IGF‐binding proteins during human osteoarthritis

A previous hypothesis stated that during osteoarthritis (OA) increased insulin‐like growth factor (IGF) binding proteins (IGFBPs) sequester IGFs and limit their access to the cell. The objective of this article was to test this by: (1) quantifying IGF and IGFBP‐3 as well as their ratios in human OA cartilages, and (2) measuring the metabolic responses of diseased cartilage to IGF‐I and its IGFBP‐insensitive analogs. Knee or hip OA cartilages were staged for OA by histology. Cartilage slices were either extracted for assays of IGF proteins, or maintained intact as organ cultures. Proteoglycan (PG) metabolism ± IGFs was measured by use of the ³⁵S‐sulfate precursor. IGFBP‐3 (ng/mg protein) was weakly correlated with OA score by regression analysis (R² = 0.122; p = 0.040; n = 35). IGF‐I (ng/mg protein) was constant across all OA groups (ANOVA; p = .428, n = 18) and the IGF‐I/IGFBP‐3 ratios were > 1 in most samples. All OA cartilages responded to hrIGF‐I by increasing PG synthesis [average 2.29‐fold ± 0.55 (±SD) at saturation, n = 12] irrespective of OA score. The des (1–3) IGF‐I analog (which lacks the three N‐terminal amino acids) had similar maximal effects (average 2.23‐fold stimulation ± 0.71, n = 10), but it was more effective in two out of three samples at suboptimal doses. The effect of hrIGF‐I, des (1–3) IGF‐I, or the B‐chain analog on degradation was minimal. In summary, catabolism was insensitive to IGF‐I, and this was probably not due to IGFBPs. By contrast, IGF‐I exerted a robust stimulation of anabolism at sufficiently high doses, even though IGFBPs could tone down the ligand effect at low doses. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:465–474, 2008     

Impact of proteoglycan‐4 and parathyroid hormone on articular cartilage

Proteoglycan‐4 (Prg4) protects synovial joints from arthropathic changes by mechanisms that are incompletely understood. Parathyroid hormone (PTH), known for its anabolic actions in bone, increases Prg4 expression and has been reported to inhibit articular cartilage degeneration in arthropathic joints. To investigate the effect of Prg4 and PTH on articular cartilage, 16‐week‐old Prg4 mutant and wild‐type mice were treated with intermittent PTH (1–34) or vehicle control daily for six weeks. Analyses included histology of the knee joint, micro‐CT of the distal femur, and serum biochemical analysis of type II collagen fragments (CTX‐II). Compared to wild‐type littermates, Prg4 mutant mice had an acellular layer of material lining the surfaces of the articular cartilage and menisci, increased articular cartilage degradation, increased serum CTX‐II concentrations, decreased articular chondrocyte apoptosis, increased synovium SDF‐1 expression, and irregularly contoured subchondral bone. PTH‐treated Prg4 mutant mice developed a secondary deposit overlaying the acellular layer of material lining the joint surfaces, but PTH‐treatment did not alter signs of articular cartilage degeneration in Prg4 mutant mice. The increased joint SDF‐1 levels and irregular subchondral bone found in Prg4 mutant mice introduce novel candidate mechanisms by which Prg4 protects articular cartilage. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 183–190, 2013     

Impact of Pregnancy‐Associated Plasma Protein‐A Deletion on the Adult Murine Skeleton

Introduction : The metalloproteinase, pregnancy‐associated plasma protein‐A (PAPP‐A) functions to enhance local insulin‐like growth factor (IGF)‐I bioavailability through cleavage of inhibitory IGF binding proteins. Because IGF‐I is an important regulator of skeletal growth and remodeling and PAPP‐A is highly expressed by osteoblastic cells, we hypothesized that, in the absence of PAPP‐A, bone physiology would be compromised because of a blunting of local IGF‐I action even in the presence of normal circulating IGF‐I levels. Materials and Methods : pQCT, μCT, histomorphometry, and mechanical strength testing were performed on bones from PAPP‐A knockout (KO) mice and wildtype (WT) littermates at 2–12 mo of age. IGF‐I levels and bone formation and resorption markers were determined in sera from these animals. Results : Volumetric BMD in PAPP‐A KO mice measured by pQCT at the femoral midshaft, which is primarily cortical bone, was 10% less than WT at 2 mo. This difference was maintained at 4, 6, and 12 mo. Cortical thickness at this site was similarly decreased. On the other hand, trabecular bone at the distal femur (pQCT) and in the tibia (μCT) showed age‐progressive decreases in bone volume fraction in PAPP‐A KO compared with WT mice. Tibial μCT indicated a 46% relative decrease in trabecular bone volume/total volume (BV/TV) and a 28% relative decrease in trabecular thickness in PAPP‐A KO compared with WT mice at 6 mo. These trabecular deficiencies in PAPP‐A KO mice corresponded to a weakening of the bone. Serum markers and bone histomorphometry indicated that the primary impact of PAPP‐A is on skeletal remodeling resulting in a state of low‐turnover osteopenia in adult PAPP‐A KO mice. Circulating IGF‐I levels were not altered in PAPP‐A KO mice. Conclusions : PAPP‐A is a bone growth regulatory factor in vivo and, in its absence, mice show skeletal insufficiency in mass, density, architecture, and strength. The data suggest a primary role for PAPP‐A in modulating local IGF bioavailability for trabecular bone remodeling.     

Articular Cartilage Repair with Autografting Under the Influence of Insulin‐Like Growth Factor‐1 in Rabbits

Insulin‐like growth factor (IGF)‐1 has been successfully demonstrated to stimulate proteoglycan synthesis, slow down its catabolism and promote cartilage formation through well defined in vitro studies. It was therefore, assumed that IGF‐1 would eventually serve to augment current cartilage repair techniques in vivo. Study was therefore, designed to determine the influence of IGF‐1 in cartilage repair with or without autografting. For this purpose articular cartilage repair model was created in the left knee of 48 New Zealand white rabbits of either sex, 6–7 months old, weighing 1–2 kg. The articular cartilage defect was created in the femoral groove of femoro‐patellar joint using hand held trephine under xylazine and ketamine anaesthesia in all the animals. The defect created was 3 mm in diameter and 2 mm in depth. For autografting, osteochondral tissues harvested from the proximal patellar groove of the femur were placed in the distal defect and vice versa. The experimental animals were divided mainly into four groups, i.e. Group A (control), Group B (autografting), Group C (control + IGF‐1) and Group D (autografting + IGF‐1). Animals of group A and B were provided only with collagen scaffolds at 10 μg/cm² whereas animals of treatment group C and D were provided with collagen scaffolds holding 30 ng/30 μl of IGF‐1 into the defect. Evaluation of cartilage repair was done on days 15, 30 and 45 after ethically killing the animals. Initially IGF‐1 had shown the tendency for either in the maintenance of autografted cartilage or helped in proliferation of chondroblast for the repair process. However, later in the process, cartilage formation apparently declined and appeared to converge to osseous tissue. Collectively, non‐responsiveness of osteoarthritic chondrocytes to IGF‐1 could be partially attributed to either increased IGF‐binding proteins in the joint space, micromovement of the graft, lack of nutrition, dose of IGF‐1 or its half life in the current study.     

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.