Jakob T. Sieker

Bio-Enhanced Repair of the Anterior Cruciate Ligament

Suture repair of the anterior cruciate ligament (ACL) has been widely abandoned in favor of ACL reconstruction, largely because of the high rates of failure and unreliability of the outcomes after suture repair. However, there have been recent basic science studies that suggest that combining a suture repair with a biological adjunct may improve the results of suture repair of the ACL, with several studies in large animal models showing equivalent strength of an ACL treated with bio-enhanced repaired to that of an ACL graft at 3, 6, and 12 months after surgery. In addition, the groups treated with bio-enhanced repair had significantly less osteoarthritis when compared with the animals undergoing ACL reconstruction. These findings have led to a renewed interest in bio-enhanced primary repair as a way to make repair of the ACL a viable option for a select group of patients in the future.

Direct bone morphogenetic protein 2 and Indian hedgehog gene transfer for articular cartilage repair using bone marrow coagulates

OBJECTIVE Bone morphogenetic protein 2 (BMP-2, encoded by BMP2) and Indian hedgehog protein (IHH, encoded by IHH) are well known regulators of chondrogenesis and chondrogenic hypertrophy. Despite being a potent chondrogenic factor BMP-2 was observed to induce chondrocyte hypertrophy in osteoarthritis (OA), growth plate cartilage and adult mesenchymal stem cells (MSCs). IHH might induce chondrogenic differentiation through different intracellular signalling pathways without inducing subsequent chondrocyte hypertrophy. The primary objective of this study is to test the efficacy of direct BMP2 and IHH gene delivery via bone marrow coagulates to influence histological repair cartilage quality in vivo. METHOD Vector-laden autologous bone marrow coagulates with 10(11) adenoviral vector particles encoding BMP2, IHH or the Green fluorescent protein (GFP) were delivered to 3.2 mm osteochondral defects in the trochlea of rabbit knees. After 13 weeks the histological repair cartilage quality was assessed using the ICRS II scoring system and the type II collagen positive area. RESULTS IHH treatment resulted in superior histological repair cartilage quality than GFP controls in all of the assessed parameters (with P < 0.05 in five of 14 assessed parameters). Results of BMP2 treatment varied substantially, including severe intralesional bone formation in two of six joints after 13 weeks. CONCLUSION IHH gene transfer is effective to improve repair cartilage quality in vivo, whereas BMP2 treatment, carried the risk intralesional bone formation. Therefore IHH protein can be considered as an attractive alternative candidate growth factor for further preclinical research and development towards improved treatments for articular cartilage defects.

Immediate Administration of Intraarticular Triamcinolone Acetonide After Joint Injury Modulates Molecular Outcomes Associated With Early Synovitis

To test whether intraarticular corticosteroid injection mitigates injury‐induced synovitis and collagen degradation after anterior cruciate ligament transection (ACLT) and to characterize the synovial response using a functional genomics approach in a preclinical model of posttraumatic osteoarthritis.

Electron beam sterilization does not have a detrimental effect on the ability of extracellular matrix scaffolds to support in vivo ligament healing

Extracellular matrix (ECM) scaffolds have been used to enhance anterior cruciate ligament (ACL) repair in large animal models. To translate this technology to clinical care, identifying a method which effectively sterilizes the material without significantly impairing in vivo function is desirable. Sixteen Yorkshire pigs underwent ACL transection and were randomly assigned to bridge‐enhanced ACL repair—primary suture repair of the ACL with addition of autologous blood soaked ECM scaffold—with either (i) an aseptically processed ECM scaffold, or (ii) an electron beam irradiated ECM scaffold. Primary outcome measures included sterility of the scaffold and biomechanical properties of the scaffold itself and the repaired ligament at 8 weeks after surgery. Scaffolds treated with 15 kGy electron beam irradiation had no bacterial or fungal growth noted, while aseptically processed scaffolds had bacterial growth in all tested samples. The mean biomechanical properties of the scaffold and healing ligament were lower in the electron beam group; however, differences were not statistically significant. Electron beam irradiation was able to effectively sterilize the scaffolds. In addition, this technique had only a minimal impact on the in vivo function of the scaffolds when used for ligament healing in the porcine model.

Effect of low-temperature ethylene oxide and electron beam sterilization on the in vitro and in vivo function of reconstituted extracellular matrix-derived scaffolds.

Reconstituted extracellular matrix (ECM)-derived scaffolds are commonly utilized in preclinical tissue engineering studies as delivery vehicles for cells and growth factors. Translation into clinical use requires identifying a sterilization method that effectively removes bacteria but does not harm scaffold function. To determine effectiveness of sterilization and impact on ECM scaffold integrity and function, low-temperature ethylene oxide and 15 kGy electron beam irradiation techniques were evaluated. Scaffold sterility was assessed in accordance to United States Pharmacopeia Chapter 71. Scaffold matrix degradation was determined in vitro using enzymatic resistance tests and gel electrophoresis. Scaffold mechanics including elastic modulus, yield stress and collapse modulus were tested. Lastly, 14 Yorkshire pigs underwent ACL transection and bio-enhanced ACL repair using sterilized scaffolds. Histologic response of ligament, synovium, and lymph nodes was compared at 4, 6, and 8 weeks. Ethylene oxide as well as electron beam irradiation yielded sterile scaffolds. Scaffold resistance to enzymatic digestion and protein integrity slightly decreased after electron beam irradiation while ethylene oxide altered scaffold matrix. Scaffold elastic modulus and yield stress were increased after electron beam treatment, while collapse modulus was increased after ethylene oxide treatment. No significant changes in ACL dimensions, in vivo scaffold resorption rate, or histologic response of synovium, ligament, and lymph nodes with either terminal sterilization technique were detectable. In conclusion, this study identifies two methods to terminally sterilize an ECM scaffold. In vitro scaffold properties were slightly changed without significantly influencing the biologic responses of the surrounding tissues in vivo. This is a critical step toward translating new tissue engineering strategies to clinical trials.

Extracellular matrix‐blood composite injection reduces post‐traumatic osteoarthritis after anterior cruciate ligament injury in the rat

The objective of this study was to determine if an injection of a novel extracellular matrix scaffold and blood composite (EMBC) after anterior cruciate ligament (ACL) injury would have a mitigating effect on post‐traumatic osteoarthritis (PTOA) development in rat knees. Lewis rats underwent unilateral ACL transection and were divided into three groups as follows: (1) no further treatment (ACLT; n = 10); (2) an intra‐articular injection of EMBC on day 0 (INJ0; n = 11); and (3) an intra‐articular injection of EMBC on day 14 (INJ14; n = 11). Ten additional animals received capsulotomy only (n = 10, SHAM group). The OARSI histology scoring of the tibial cartilage and micro‐CT of the tibial epiphysis were performed after 35 days. The ratio of intact/treated hind limb forces during gait was determined using a variable resistor walkway. The OARSI cartilage degradation sum score and total degeneration width were significantly greater in the ACLT group when compared to the INJ0 (p = 0.031, and p = 0.005) and INJ14 (p = 0.022 and p = 0.04) group. Weight bearing on the operated limb only decreased significantly in the ACLT group (p = 0.048). In the rat ACL transection model, early or delayed injection of EMBC ameliorated the significant decrease in weight bearing and cartilage degradation seen in knees subjected to ACL transection without injection. The results indicate that the injection of EMBC may slow the process of PTOA following ACL injury and may provide a promising treatment for PTOA.

Transcriptional Profiling of Articular Cartilage in a Porcine Model of Early Post-traumatic Osteoarthritis†

To identify the molecular pathophysiology present in early post‐traumatic osteoarthritis (PTOA), the transcriptional profile of articular cartilage and its response to surgical PTOA induction were determined. Thirty six Yucatan minipigs underwent anterior cruciate ligament (ACL) transection and were randomly assigned in equal numbers to no further treatment, reconstruction or ligament repair. Cartilage was harvested at 1 and 4 weeks post‐operatively and histology and RNA‐sequencing were performed and compared to controls. Microscopic cartilage scores significantly worsened at 1 (p = 0.028) and 4 weeks (p = 0.001) post‐surgery relative to controls, but did not differ between untreated, reconstruction or repair groups. Gene expression after ACL reconstruction and ACL transection were similar, with only 0.03% (including SERPINB7 and CR2) and 0.2% of transcripts (including INHBA) differentially expressed at 1 and 4 weeks respectively. COL2A1, COMP, SPARC, CHAD, and EF1ALPHA were the most highly expressed non ribosomal, non mitochondrial genes in the controls and remained abundant after surgery. A total of 1,275 genes were differentially expressed between 1 and 4 weeks post‐surgery. With the treatment groups pooled, 682 genes were differentially expressed at both time‐points, with the most significant changes observed in MMP1, COCH, POSTN, CYTL1, and PTGFR. This study confirmed the development of a microscopic PTOA stage after ACL surgery in the porcine model. Upregulation of multiple proteases (including MMP1 and ADAMTS4) were found; however, the level of expression remained orders of magnitude below that of extracellular matrix protein‐coding genes (including COL2A1 and ACAN). In summary, genes with established roles in PTOA as well as novel targets for specific intervention were identified.

Effective repair of joint cartilage using human pluripotent stem cell-derived tissue

Adult articular cartilage lacks significant regenerative capacity, and damage to this tissue often leads to progressive joint degeneration (osteoarthritis). We developed strategies to generate articular cartilage from human pluripotent stem cells (hPSCs) as a source of clinically relevant tissues for joint repair1. Previously, we demonstrated that these chondrocytes retain cartilage forming potential following subcutaneous implantation in mice. In this report, we evaluated the potential of human embryonic stem cell (hESC)-derived articular cartilage tissue to repair osteochondral defects created in the rat knee. Following implantation, the hESCderived cartilage maintained a proteoglycan and type II collagen-rich matrix, and was well integrated with native rat tissue at the basal and lateral surfaces. The ability to generate cartilage tissue with integrative and reparative properties from an unlimited and robust cell source represents a significant clinical advance for cartilage repair that can be applied to large animal models and ultimately to patient care.

Mesenchymal Stem Cells Isolated from the Anterior Cruciate Ligament: Characterization and Comparison of Cells from Young and Old Donors

Purpose Mesenchymal stem cells (MSCs) isolated from the anterior cruciate ligament (ACL) share multiple characteristics of bone marrow-derived mesenchymal stem cells (BMSCs), allowing their use for regenerative therapies. Injuries to the ACL can affect people of all ages. This study assesses whether the regenerative potential of ACL-derived MSCs (ACL-MSCs) from old donors is as high as the potential of ACL-MSCs from young donors. Materials and Methods ACL-MSCs were isolated from ACL tissues obtained from young and old donors at the time of ACL reconstruction or arthroplasty. Proliferative capacity, multilineage differentiation potential (chondrogenic, osteogenic, and adipogenic lineages), and transcriptome-wide gene expression were assessed and compared between young and old donors. BMSCs of middle-aged donors served as an additional comparator. Results No substantial differences between ACL-MSCs from young and old donors were observed in their proliferative capacity and multilineage differentiation potential. The latter did not substantially differ between both ACL-MSC groups and BMSCs. Differential expression of genes related to the cytoskeleton and to protein dephosphorylation amongst other pathways was detected between ACL-MSCs from young and old donors. Conclusions Regenerative potential of ACL-MSCs from old donors was not substantially lower than that from young donors, suggesting that regenerative therapies of ACL tears are feasible in both age groups. In vivo studies of the effect of age on the efficacy of such therapies are needed.

Transcriptional profiling of synovium in a porcine model of early post-traumatic osteoarthritis: TRANSCRIPTIONAL PROFILING OF SYNOVIUM

To determine the transcriptional profile of synovium during the molecular phase of post‐traumatic osteoarthritis, anterior cruciate ligament transections (ACL) were performed in 36 Yucatan minipigs. Equal numbers were randomly assigned to no further treatment, ACL reconstruction or repair. Perimeniscal synovium for histopathology and RNA‐sequencing was harvested at 1 and 4 weeks post‐operatively and from six healthy control animals. Microscopic synovitis scores significantly worsened at 1 (p < 0.001) and 4 weeks (p = 0.003) post‐surgery relative to controls, and were driven by intimal hyperplasia and increased stromal cellularity without inflammatory infiltrates. Synovitis scores were similar between no treatment, reconstruction, and repair groups (p ≥ 0.668). Relative to no treatment at 1 week, 88 and 367 genes were differentially expressed in the reconstruction and repair groups, respectively (227 and 277 at 4 weeks). Relative to controls and with the treatment groups pooled, 1,683 transcripts were concordantly differentially expressed throughout the post‐surgery time‐course. Affected pathways included, proteolysis_connective tissue degradation (including upregulations of protease‐encoding MMP1, MMP13, and ADAMTS4), and development_cartilage development (including upregulations of ACAN, SOX9, and RUNX2), among others. Using linear regression, significant associations of post‐surgery synovial expression levels of 20 genes with the articular cartilage glycosaminoglycan loss were identified. These genes were predominantly related to embryonic skeletal system development and included RUNX2. In conclusion, this study confirmed an increased synovial expression of genes that may serve as targets to prevent cartilage degradation, including MMP1, MMP13, and ADAMTS4, in knees with microscopic synovitis and cartilage proteoglycan loss. Attractive novel targets include regulators of embryonic developmental processes in synovium.