Alan J. Nixon

Insulin-like growth factor-I enhances cell-based repair of articular cartilage

Composites of chondrocytes and polymerised fibrin were supplemented with insulin-like growth factor-I (IGF-I) during the arthroscopic repair of full-thickness cartilage defects in a model of extensive loss of cartilage in horses. Repairs facilitated with IGF-I and chondrocyte-fibrin composites, or control defects treated with chondrocyte-fibrin composites alone, were compared before death by the clinical appearance and repeated analysis of synovial fluid, and at termination eight months after surgery by tissue morphology, collagen typing, and biochemical assays. The structure of cartilage was evaluated histologically by Toluidine Blue reaction and collagen type-I and type-II in situ hybridisation and immunohistochemistry. Repair tissue was biochemically evaluated by DNA assay, proteoglycan quantitation and characterisation, assessment of collagen by reverse-phase high-performance liquid chromatography, and collagen typing using cyanogen bromide digestion and peptide separation by polyacrylamide gel electrophoresis. The results at eight months showed that the addition of IGF-I to chondrocyte grafts enhanced chondrogenesis in cartilage defects, including incorporation into surrounding cartilage. Gross filling of defects was improved, and the tissue contained a higher proportion of cells producing type-II collagen. Measurements of collagen type II showed improved levels in IGF-I-treated defects, supporting in situ hybridisation and immunohistochemical assessments of the defects. IGF-I improves the repair capabilities of chondrocyte-fibrin grafts in large full-thickness repair models.

Chondrocytic differentiation of mesenchymal stem cells sequentially exposed to transforming growth factor‐β1 in monolayer and insulin‐like growth factor‐I in a three‐dimensional matrix

This study evaluated chondrogenesis of mesenchymal progenitor stem cells (MSCs) cultured initially under pre‐confluent monolayer conditions exposed to transforming growth factor‐β (TGF‐β), and subsequently in three‐dimensional cultures containing insulin‐like growth factor I (IGF‐I). Bone marrow aspirates and chondrocytes were obtained from horses and cultured in monolayer with 0 or 5 ng of TGF‐β1 per ml of medium for 6 days. TGF‐β1 treated and untreated cultures were distributed to three‐dimensional fibrin disks containing 0 or 100 ng of IGF‐I per ml of medium to establish four treatment groups. After 13 days, cultures were assessed by toluidine blue staining, collagen types I and II in situ hybridization and immunohistochemistry, proteoglycan production by [Chondrocytic differentiation of mesenchymal stem cells sequentially exposed to transforming growth factor‐β1 in monolayer and insulin‐like growth factor‐I in a three‐dimensional matrix35S]‐sulfate incorporation, and disk DNA content by fluorometry. Mesenchymal cells in monolayer cultures treated with TGF‐β1 actively proliferated for the first 4 days, developed cellular rounding, and formed cell clusters. Treated MSC cultures had a two‐fold increase in medium proteoglycan content. Pretreatment of MSCs with TGF‐β1 followed by exposure of cells to IGF‐I in three‐dimensional culture significantly increased the formation of markers of chondrocytic function including disk proteoglycan content and procollagen type II mRNA production. However, proteoglycan and procollagen type II production by MSCs remained lower than parallel chondrocyte cultures. MSC pretreatment with TGF‐β1 without sequential IGF‐I was less effective in initiating expression of markers of chondrogenesis. This study indicates that although MSC differentiation was less than complete when compared to mature chondrocytes, chondrogenesis was observed in IGF‐I supplemented cultures, particularly when used in concert with TGF‐β1 pretreatment.

Concentrated Bone Marrow Aspirate Improves Full-thickness Cartilage Repair Compared with Microfracture in the Equine Model

BACKGROUND The purpose of this study was to compare the outcomes of treatment with bone marrow aspirate concentrate, a simple, one-step, autogenous, and arthroscopically applicable method, with the outcomes of microfracture with regard to the repair of full-thickness cartilage defects in an equine model. METHODS Extensive (15-mm-diameter) full-thickness cartilage defects were created on the lateral trochlear ridge of the femur in twelve horses. Bone marrow was aspirated from the sternum and centrifuged to generate the bone marrow concentrate. The defects were treated with bone marrow concentrate and microfracture or with microfracture alone. Second-look arthroscopy was performed at three months, and the horses were killed at eight months. Repair was assessed with use of macroscopic and histological scoring systems as well as with quantitative magnetic resonance imaging. RESULTS No adverse reactions due to the microfracture or the bone marrow concentrate were observed. At eight months, macroscopic scores (mean and standard error of the mean, 9.4 + or - 1.2 compared with 4.4 + or - 1.2; p = 0.009) and histological scores (11.1 + or - 1.6 compared with 6.4 + or - 1.2; p = 0.02) indicated improvement in the repair tissue in the bone marrow concentrate group compared with that in the microfracture group. All scoring systems and magnetic resonance imaging data indicated that delivery of the bone marrow concentrate resulted in increased fill of the defects and improved integration of repair tissue into surrounding normal cartilage. In addition, there was greater type-II collagen content and improved orientation of the collagen as well as significantly more glycosaminoglycan in the bone marrow concentrate-treated defects than in the microfracture-treated defects. CONCLUSIONS Delivery of bone marrow concentrate can result in healing of acute full-thickness cartilage defects that is superior to that after microfracture alone in an equine model. CLINICAL RELEVANCE Delivery of bone marrow concentrate to cartilage defects has the clinical potential to improve cartilage healing, providing a simple, cost-effective, arthroscopically applicable, and clinically effective approach for cartilage repair.

EFFECT OF ADIPOSE-DERIVED NUCLEATED CELL FRACTIONS ON TENDON REPAIR IN HORSES WITH COLLAGENASE-INDUCED TENDINITIS

OBJECTIVE To assess the potential of adipose-derived nucleated cell (ADNC) fractions to improve tendon repair in horses with collagenase-induced tendinitis. ANIMALS 8 horses. PROCEDURES Collagenase was used to induce tendinitis in the superficial digital flexor tendon of 1 forelimb in each horse. Four horses were treated by injection of autogenous ADNC fractions, and 4 control horses were injected with PBS solution. Healing was compared by weekly ultrasonographic evaluation. Horses were euthanatized at 6 weeks. Gross and histologic evaluation of tendon structure, fiber alignment, and collagen typing were used to define tendon architecture. Biochemical and molecular analyses of collagen, DNA, and proteoglycan and gene expression of collagen type I and type III, decorin, cartilage oligomeric matrix protein (COMP), and insulin-like growth factor-I were performed. RESULTS Ultrasonography revealed no difference in rate or quality of repair between groups. Histologic evaluation revealed a significant improvement in tendon fiber architecture; reductions in vascularity, inflammatory cell infiltrate, and collagen type III formation; and improvements in tendon fiber density and alignment in ADNC-treated tendons. Repair sites did not differ in DNA, proteoglycan, or total collagen content. Gene expression of collagen type I and type III in treated and control tendons were similar. Gene expression of COMP was significantly increased in ADNC-injected tendons. CONCLUSIONS AND CLINICAL RELEVANCE ADNC injection improved tendon organization in treated tendons. Although biochemical and molecular differences were less profound, tendons appeared architecturally improved after ADNC injection, which was corroborated by improved tendon COMP expression. Use of ADNC in horses with tendinitis appears warranted.

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.

Insulin-like growth factor-I improves cellular and molecular aspects of healing in a collagenase-induced model of flexor tendinitis

Flexor tendinitis is a common and debilitating injury of elite and recreational athletes. Healing may be improved through intratendinous injection of insulin‐like growth factor‐I (IGF‐I), which has been shown in vitro to stimulate mitogenesis and enhance tendon matrix production. This study investigated the effects of intratendinous injection of IGF‐I on tendon healing in an equine model of flexor tendinitis. Collagenase‐induced lesions were created in the tensile region of the flexor digitorum superficialis tendon of both forelimbs of eight horses. Treated tendons were injected with 2 μg rhIGF‐I intralesionally every other day for 10 injections, while controls received 0.9% NaCl. Tendon fiber deposition and organization were evaluated serially using ultrasonography throughout the 8 week trial period. Following euthanasia, the tendons were harvested and DNA, hydroxyproline, and glycosaminoglycan content determined, mechanical strength and stiffness evaluated, gene expression and spatial arrangement of collagen types I and III assessed by northern blot and in situ hybridization, and tendon fiber architecture assessed by polarized light microscopy. Local soft tissue swelling was reduced in the IGF‐I treated limbs. Similarly, lesion size in IGF‐I treated tendons was smaller 3 and 4 weeks after initiation of treatment. Cell proliferation and collagen content of the IGF‐I treated tendons were increased compared to controls. Mechanically, IGF‐I treated tendons showed a trend toward increased stiffness compared to saline treated controls. Considered together with the decreased soft tissue swelling and improved sonographic healing, these data support the potential use of intralesional IGF‐I for treatment of debilitating tendon injuries.

Acceleration of cartilage repair by genetically modified chondrocytes over expressing bone morphogenetic protein-7

Background: Cartilage has a limited capacity to heal. Although chondrocyte transplantation is a useful therapeutic strategy, the repair process can be lengthy. Previously we have shown that over expression of bone morphogenetic protein‐7 (BMP‐7) in chondrocytes by adenovirus‐mediated gene transfer leads to increased matrix synthesis and cartilage‐like tissue formation in vitro. In this context we hypothesized that implantation of genetically modified chondrocytes expressing BMP‐7 would accelerate the formation of hyaline‐like repair tissue in an equine model of cartilage defect repair.

Chondrocyte-laden collagen scaffolds for resurfacing extensive articular cartilage defects.

Chondrocyte-collagen composites were evaluated for resurfacing of large articular defects. Isolated chondrocytes were cultured in expanded collagen scaffolds for 7-10 days to provide a composite containing 3.6 x 10(4) cells/mm3. The graft was transplanted into 15 mm full thickness articular defects in the femoropatellar joint of 12 horses using arthroscopic techniques. Ungrafted defects in the opposite femoropatellar joint served as controls. Synovial fluid, clinical progress and pain responses were evaluated in groups of 6 horses over 4-month and 8-month periods. Following termination, gross, histochemical and histologic evaluations of the repair tissues and synovial membrane were performed. Arthroscopic defect debridement and chondrocyte implantation resulted in minimal post-operative effusion or pain, and synovial fluid constituents were not significantly different in grafted and ungrafted joints. Gross differences in grafted defects were not apparent. Increased chondrocyte numbers and cartilage histochemical staining were evident in the deeper layers of grafted defects, whereas ungrafted defects were almost entirely fibrous tissue. The surface layers of grafted defects were fibrous tissue. There were no synovial fluid cellular responses, synovial membrane histiocytic reaction or subchondral bone infiltrates to suggest immune-related reaction to the allograft cells. Chondrocyte-collagen grafts were arthroscopically implanted and resulted in improved cartilage healing in extensive defects. However, the structural organization of the surface layers was inadequate and suggested poor long-term durability.

Genetic modification of chondrocytes with insulin-like growth factor-1 enhances cartilage healing in an equine model

Gene therapy with insulin-like growth factor-1 (IGF-1) increases matrix production and enhances chondrocyte proliferation and survival in vitro. The purpose of this study was to determine whether arthroscopically-grafted chondrocytes genetically modified by an adenovirus vector encoding equine IGF-1 (AdIGF-1) would have a beneficial effect on cartilage healing in an equine femoropatellar joint model. A total of 16 horses underwent arthroscopic repair of a single 15 mm cartilage defect in each femoropatellar joint. One joint received 2 x 10(7) AdIGF-1 modified chondrocytes and the contralateral joint received 2 x 10(7) naive (unmodified) chondrocytes. Repairs were analysed at four weeks, nine weeks and eight months after surgery. Morphological and histological appearance, IGF-1 and collagen type II gene expression (polymerase chain reaction, in situ hybridisation and immunohistochemistry), collagen type II content (cyanogen bromide and sodium dodecyl sulphate-polyacrylamide gel electrophoresis), proteoglycan content (dimethylmethylene blue assay), and gene expression for collagen type I, matrix metalloproteinase (MMP)-1, MMP-3, MMP-13, aggrecanase-1, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and TIMP-3 were evaluated. Genetic modification of chondrocytes significantly increased IGF-1 mRNA and ligand production in repair tissue for up to nine weeks following transplantation. The gross and histological appearance of IGF-1 modified repair tissue was improved over control defects. Gross filling of defects was significantly improved at four weeks, and a more hyaline-like tissue covered the lesions at eight months. Histological outcome at four and nine weeks post-transplantation revealed greater tissue filling of defects transplanted with genetically modified chondrocytes, whereas repair tissue in control defects was thin and irregular and more fibrous. Collagen type II expression in IGF-1 gene-transduced defects was increased 100-fold at four weeks and correlated with increased collagen type II immunoreaction up to eight months. Genetic modification of chondrocytes with AdIGF-1 prior to transplantation improved early (four to nine weeks), and to a lesser degree long-term, cartilage healing in the equine model. The equine model of cartilage healing closely resembles human clinical cartilage repair. The results of this study suggest that cartilage healing can be enhanced through genetic modification of chondrocytes prior to transplantation.

Gene-based approaches for the repair of articular cartilage.

Gene transfer technology has opened novel treatment avenues toward the treatment of damaged musculoskeletal tissues, and may be particularly beneficial to articular cartilage. There is no natural repair mechanism to heal damaged or diseased cartilage. Existing pharmacologic, surgical and cell based treatments may offer temporary relief but are incapable of restoring damaged cartilage to its normal phenotype. Gene transfer provides the capability to achieve sustained, localized presentation of bioactive proteins or gene products to sites of tissue damage. A variety of cDNAs have been cloned which may be used to stimulate biological processes that could improve cartilage healing by (1) inducing mitosis and the synthesis and deposition of cartilage extracellular matrix components by chondrocytes, (2) induction of chondrogenesis by mesenchymal progenitor cells, or (3) inhibiting cellular responses to inflammatory stimuli. The challenge is to adapt this technology into a useful clinical treatment modality. Using different marker genes, the principle of gene delivery to synovium, chondrocytes and mesenchymal progenitor cells has been convincingly demonstrated. Following this, research efforts have begun to move to functional studies. This involves the identification of appropriate gene or gene combinations, incorporation of these cDNAs into appropriate vectors and delivery to specific target cells within the proper biological context to achieve a meaningful therapeutic response. Methods currently being explored range from those as simple as direct delivery of a vector to a cartilage defect, to synthesis of cartilaginous implants through gene-enhanced tissue engineering. Data from recent efficacy studies provide optimism that gene delivery can be harnessed to guide biological processes toward both accelerated and improved articular cartilage repair.