Loss of periostin function impairs ligament fibroblast activity and facilitates ROS-mediated cellular senescence.

Anterior cruciate ligament (ACL) injuries pose a significant challenge due to their limited healing potential, often resulting in premature arthritis. The factors and mechanisms contributing to this inadequate healing process remain elusive. During the acute phase of injury, ACL tissues express elevated periostin levels that decline over time. The functional significance of periostin in ligament biology remains understudied. In this study, we investigated the functional and mechanistic implications of periostin deficiency in ACL biology, utilizing ligament fibroblasts derived from patients and a murine model of ACL rupture. Our investigations unveiled that periostin knockdown compromised fibroblast growth characteristics, hindered the egress of progenitor cells from explants, and arrested cell-cycle progression, resulting in the accumulation of cells in the G0/G1 phase and moderate apoptosis. Concurrently, a significant reduction in the expression of cell-cycle and matrix-related genes was observed. Moreover, periostin deficiency triggered apoptosis through STAT3Y705/p38MAPK signaling and induced cellular senescence through increased production of reactive oxygen species (ROS). Mechanistically, inhibition of ROS production mitigated cell senescence in these cells. Notably, in vivo data revealed that ACL in Postn-/- mice exhibited a higher tearing frequency than wild-type mice under equivalent loading conditions. Furthermore, injured ACL with silenced periostin expression, achieved through nanoparticle-siRNA complex delivery, displayed an elevated propensity for apoptosis and senescence compared to intact ACL in C57BL/6 mice. Together, our findings underscore the pivotal role of periostin in ACL health, injury, and potential for healing.

Bone marrow stromal and anterior cruciate ligament remnant cell co-culture-derived extracellular vesicles promote cell activity in both cell types.

The significance of anterior cruciate ligament (ACL) remnants during reconstruction remains unclear. Co-culturing ACL remnant cells and bone marrow stromal cells (BMSCs) may reduce apoptosis and enhance hamstring tendon activity. This study investigated whether extracellular vesicles (EVs), which facilitate cell-cell interactions, act as the active components, improving graft maturation in this co-culture. The effects of EVs on cell viability, proliferation, migration and gene expression in the rabbit ACL remnant cells and BMSCs were assessed using control (BMSC-only culture), co-culture (ACL remnant cells and BMSCs, CM) and co-culture without EVs (CM ∆ EVs) media. EVs were isolated from control (BMSC-EV) and co-culture (CM-EV) media and characterized. CM significantly enhanced the proliferation, migration and expression of transforming growth factor (TGF-ß)-, vascular endothelial growth factor (VEGF)-, collagen synthesis- and tenogenesis-related genes. However, CM-induced effects were reversed by the CM ∆ EVs treatment. CM-EV treatment exhibited higher potential to enhance proliferation, migration and gene expression in the ACL remnant cells and BMSCs than BMSC-EV and non-EV treatments. In conclusion, EVs, secreted under the coexistence of ACL remnant cells and BMSCs, primarily increase the cell viability, proliferation, migration and gene expression of collagen synthesis-, TGF-ß-, VEGF- and tenogenesis-related genes in both cell types.

Commitment of human mesenchymal stromal cells towards ACL fibroblast differentiation upon rAAV-mediated FGF-2 and TGF-ß overexpression using pNaSS-grafted PCL films.

Despite various clinical options, human anterior cruciate ligament (ACL) lesions do not fully heal. Biomaterial-guided gene therapy using recombinant adeno-associated virus (rAAV) vectors may improve the intrinsic mechanisms of ACL repair. Here, we examined whether poly(sodium styrene sulfonate)-grafted poly(ε-caprolactone) (pNaSS-grafted PCL) films can deliver rAAV vectors coding for the reparative basic fibroblast growth factor (FGF-2) and transforming growth factor beta (TGF-ß) in human mesenchymal stromal cells (hMSCs) as a source of implantable cells in ACL lesions. Efficient and sustained rAAV-mediated reporter (red fluorescent protein) and therapeutic (FGF-2 and TGF-ß) gene overexpression was achieved in the cells for at least 21 days in particular with pNaSS-grafted PCL films relative to all other conditions (up to 5.2-fold difference). Expression of FGF-2 and TGF-ß mediated by rAAV using PCL films increased the levels of cell proliferation, the DNA contents, and the deposition of proteoglycans and of type-I and -III collagen (up to 2.9-fold difference) over time in the cells with higher levels of transcription factor expression (Mohawk, Scleraxis) (up to 1.9-fold difference), without activation of inflammatory tumor necrosis alpha especially when using pNaSS-grafted PCL films compared with the controls. Overall, the effects mediated by TGF-ß were higher than those promoted by FGF-2, possibly due to higher levels of gene expression achieved upon rAAV gene transfer. This study shows the potential of using functionalized PCL films to apply rAAV vectors for ACL repair.

Increased vascular endothelial growth factor expression is associated with cruciate ligament degeneration in patients with osteoarthritis of the knee.

BACKGROUND: This study aimed to investigate the expression of vascular endothelial growth factor (VEGF) in cruciate ligaments from patients with osteoarthritis (OA). It was hypothesized that the expression level of VEGF is associated with the extent of degeneration of the cruciate ligaments. METHODS: Remnants of anterior cruciate ligaments (ACLs) from patients with acute ACL injury due to trauma, and ACLs and posterior cruciate ligaments (PCLs) from patients with primary OA were assessed histologically. Samples were immunohistochemically stained with VEGF and tenomodulin, and immunopositive cells were quantitatively assessed by the histological grades of ligament degeneration. RESULTS: Histological analysis showed significant degeneration of the ACLs from OA patients compared with trauma patients, with increased expression of VEGF correlating with higher grades of degeneration. Conversely, tenomodulin expression was lower in more degenerated cruciate ligaments. The percentage of VEGF-positive cells was correlated inversely with that of tenomodulin-positive cells. CONCLUSIONS: Increased VEGF expression is associated with degeneration of cruciate ligaments in patients with osteoarthritis of the knee.

BMP signaling: A significant player and therapeutic target for osteoarthritis.

OBJECTIVE: To explore the significance of BMP signaling in osteoarthritis (OA) etiology, and thereafter propose a disease-modifying therapy for OA. METHODS: To examine the role of the BMP signaling in pathogenesis of OA, an Anterior Cruciate Ligament Transection (ACLT) surgery was performed to incite OA in C57BL/6J mouse line at postnatal day 120 (P120). Thereafter, to investigate whether activation of BMP signaling is necessary and sufficient to induce OA, we have used conditional gain- and loss-of-function mouse lines in which BMP signaling can be activated or depleted, respectively, upon intraperitoneal injection of tamoxifen. Finally, we locally inhibited BMP signaling through intra-articular injection of LDN-193189 pre- and post-onset surgically induced OA. The majority of the investigation has been conducted using micro-CT, histological staining, and immuno histochemistry to assess the disease etiology. RESULTS: Upon induction of OA, depletion of SMURF1-an intra-cellular BMP signaling inhibitor in articular cartilage coincided with the activation of BMP signaling, as measured by pSMAD1/5/9 expression. In mouse articular cartilage, the BMP gain-of-function mutation is sufficient to induce OA even without surgery. Further, genetic, or pharmacological BMP signaling suppression also prevented pathogenesis of OA. Interestingly, inflammatory indicators were also significantly reduced upon LDN-193189 intra-articular injection which inhibited BMP signaling and slowed OA progression post onset. CONCLUSION: Our findings showed that BMP signaling is crucial to the etiology of OA and inhibiting BMP signaling locally can be a potent strategy for alleviating OA.

Diterbutyl phthalate attenuates osteoarthritis in ACLT mice via suppressing ERK/c-fos/NFATc1 pathway, and subsequently inhibiting subchondral osteoclast fusion.

Osteoarthritis (OA) is the most common arthritis with a rapidly increasing prevalence. Disease progression is irreversible, and there is no curative therapy available. During OA onset, abnormal mechanical loading leads to excessive osteoclastogenesis and bone resorption in subchondral bone, causing a rapid subchondral bone turnover, cyst formation, sclerosis, and finally, articular cartilage degeneration. Moreover, osteoclast-mediated angiogenesis and sensory innervation in subchondral bone result in abnormal vascularization and OA pain. The traditional Chinese medicine Panax notoginseng (PN; Sanqi) has long been used in treatment of bone diseases including osteoporosis, bone fracture, and OA. In this study we established two-dimensional/bone marrow mononuclear cell/cell membrane chromatography/time of flight mass spectrometry (2D/BMMC/CMC/TOFMS) technique and discovered that diterbutyl phthalate (DP) was the active constituent in PN inhibiting osteoclastogenesis. Then we explored the therapeutic effect of DP in an OA mouse model with anterior cruciate ligament transaction (ACLT). After ACLT was conducted, the mice received DP (5 mg·kg-1·d-1, ip) for 8 weeks. Whole knee joint tissues of the right limb were harvested at weeks 2, 4, and 8 for analysis. We showed that DP administration impeded overactivated osteoclastogenesis in subchondral bone and ameliorated articular cartilage deterioration. DP administration blunted aberrant H-type vessel formation in subchondral bone marrow and alleviated OA pain assessed in Von Frey test and thermal plantar test. In RANKL-induced RAW264.7 cells in vitro, DP (20 µM) retarded osteoclastogenesis by suppressing osteoclast fusion through inhibition of the ERK/c-fos/NFATc1 pathway. DP treatment also downregulated the expression of dendritic cell-specific transmembrane protein (DC-STAMP) and d2 isoform of the vacuolar (H+) ATPase V0 domain (Atp6v0d2) in the cells. In conclusion, we demonstrate that DP prevents OA progression by inhibiting abnormal osteoclastogenesis and associated angiogenesis and neurogenesis in subchondral bone.

Minispheroids as a Tool for Ligament Tissue Engineering: Do the Self-Assembly Techniques and Spheroid Dimensions Influence the Cruciate Ligamentocyte Phenotype?

Spheroid culture might stabilize the ligamentocyte phenotype. Therefore, the phenotype of lapine cruciate ligamentocyte (L-CLs) minispheroids prepared either by hanging drop (HD) method or by using a novel spheroid plate (SP) and the option of methyl cellulose (MC) for tuning spheroid formation was tested. A total of 250 and 1000 L-CLs per spheroid were seeded as HDs or on an SP before performing cell viability assay, morphometry, gene expression (qRT-PCR) and protein immunolocalization after 7 (HD/SP) and 14 (SP) days. Stable and viable spheroids of both sizes could be produced with both methods, but more rapidly with SP. MC accelerated the formation of round spheroids (HD). Their circular areas decreased significantly during culturing. After 7 days, the diameters of HD-derived spheroids were significantly larger compared to those harvested from the SP, with a tendency of lower circularity suggesting an ellipsoid shape. Gene expression of decorin increased significantly after 7 days (HD, similar trend in SP), tenascin C tended to increase after 7 (HD/SP) and 14 days (SP), whereas collagen type 1 decreased (HD/SP) compared to the monolayer control. The cruciate ligament extracellular matrix components could be localized in all mini-spheroids, confirming their conserved expression profile and their suitability for ligament tissue engineering.

Chondroprotective Effects of a Histone Deacetylase Inhibitor, Panobinostat, on Pain Behavior and Cartilage Degradation in Anterior Cruciate Ligament Transection-Induced Experimental Osteoarthritic Rats.

Osteoarthritis (OA) is the most common articular degenerative disease characterized by chronic pain, joint inflammation, and movement limitations, which are significantly influenced by aberrant epigenetic modifications of numerous OA-susceptible genes. Recent studies revealed that both the abnormal activation and differential expression of histone deacetylases (HDACs) might contribute to OA pathogenesis. In this study, we investigated the chondroprotective effects of a marine-derived HDAC inhibitor, panobinostat, on anterior cruciate ligament transection (ACLT)-induced experimental OA rats. The intra-articular administration of 2 or 10 µg of panobinostat (each group, n = 7) per week from the 6th to 17th week attenuates ACLT-induced nociceptive behaviors, including secondary mechanical allodynia and weight-bearing distribution. Histopathological and microcomputed tomography analysis showed that panobinostat significantly prevents cartilage degeneration after ACLT. Moreover, intra-articular panobinostat exerts hypertrophic effects in the chondrocytes of articular cartilage by regulating the protein expressions of HDAC4, HDAC6, HDAC7, runt-domain transcription factor-2, and matrix metalloproteinase-13. The study indicated that HDACs might have different modulations on the chondrocyte phenotype in the early stages of OA development. These results provide new evidence that panobinostat may be a potential therapeutic drug for OA.

Distinct expression pattern of periostin splice variants in chondrocytes and ligament progenitor cells.

Periostin (POSTN), a secretory matricellular matrix protein, plays a multitude of biologic functions. Various splice variants of POSTN have been described; however, their expression pattern and functional implications are not completely understood. This study was undertaken to decipher the differential expression pattern of POSTN and its splice variants in various tissues and cell types. We show that POSTN was more highly expressed in anterior cruciate ligament (ACL) remnants compared with articular cartilage at the cellular and tissue level. Isoforms 1 and 8 were highly expressed only in articular chondrocytes, suggesting their splice-specific regulation in chondrocytes. To discern the role of total POSTN and full-length human POSTN isoform 1 (hPOSTN-001), we stably transfected human chondrosarcoma 1 (hCh-1) cell line with hPOSTN-001 using a pcDNA3.1-hPOSTN-001 construct. RNA-sequencing analysis of hCh-1 cells identified differentially expressed genes with a known role in chondrocyte function and osteoarthritis. Similar expression of a subset of candidate genes was revealed in ACL progenitor cells and chondrocytes as well as in ACL progenitor cells in which POSTN activity was altered by overexpression and by small interfering RNA gene knockdown. Cells expressing total POSTN, not isoform 1, exhibited increased cell adhesion potential. These findings suggest an important role for POSTN in the knee.-Cai, L., Brophy, R. H., Tycksen, E. D., Duan, X., Nunley, R. M., Rai, M. F. Distinct expression pattern of periostin splice variants in chondrocytes and ligament progenitor cells.

SV40 Transfected Human Anterior Cruciate Ligament Derived Ligamentocytes-Suitable as a Human in Vitro Model for Ligament Reconstruction?

Cultured human primary cells have a limited lifespan undergoing dedifferentiation or senescence. Anterior cruciate ligaments (ACL) are hypocellular but tissue engineering (TE) requires high cell numbers. Simian virus (SV) 40 tumor (T) antigen expression could extend the lifespan of cells. This study aimed to identify cellular changes induced by SV40 expression in human ACL ligamentocytes by comparing them with non-transfected ligamentocytes and tissue of the same donor to assess their applicability as TE model. Human ACL ligamentocytes (40-year-old female donor after ACL rupture) were either transfected with a SV40 plasmid or remained non-transfected (control) before monitored for SV40 expression, survival, and DNA content. Protein expression of cultured ligamentocytes was compared with the donor tissue. Ligamentocyte spheroids were seeded on scaffolds embroidered either from polylactic acid (PLA) threads solely or combined PLA and poly (L-lactide-co-ε-caprolactone) (P(LA-CL)) threads. These scaffolds were further functionalized with fluorination and fibrillated collagen foam. Cell distribution and survival were monitored for up to five weeks. The transfected cells expressed the SV40 antigen throughout the entire observation time, but often exhibited random and incomplete cell divisions with significantly more dying cells, significantly more DNA and more numerous nucleoli than controls. The expression profile of non-transfected and SV40-positive ligamentocytes was similar. In contrast to controls, SV40-positive cells formed larger spheroids, produced less vimentin and focal adhesions and died on the scaffolds after 21 d. Functionalized scaffolds supported human ligamentocyte growth. SV40 antigen expressing ligamentocytes share many properties with their non-transfected counterparts suggesting them as a model, however, applicability for TE is limited.

Resistin concentrations in serum and stifle synovial fluid from normal and cruciate deficient dogs with osteoarthritis.

OBJECTIVE: To compare synovial fluid (SF) resistin concentrations in healthy dogs to dogs with osteoarthritis (OA) secondary to cranial cruciate ligament (CrCL) injury and to correlate resistin concentrations with body condition score (BCS) and evaluate resistin release from peripheral blood mononuclear cells (PBMC) and adipocytes. STUDY DESIGN: Controlled, prospective, clinical study ANIMALS: Thirty-nine client-owned dogs, 13 healthy and 26 with secondary OA, were enrolled. Blood was collected from six healthy purpose-bred dogs for PBMC culture. An additional six mixed-breed dogs were used for adipocyte collection and culture. METHODS: Resistin concentrations were measured with a canine-specific enzyme-linked immunoabsorbent assay. Resistin was compared between healthy SF and OA SF with Student's t test. Correlation of resistin concentrations to BCS was performed. Peripheral blood mononuclear cells and adipocytes were cultured under three conditions: negative control, lipopolysaccharide, and concanavalin A (Con A). A linear mixed model was used to determine differences in resistin concentrations among treatments. RESULTS: Resistin concentrations in OA SF were comparable to healthy SF. Neither serum nor SF resistin was correlated with BCS. Cultured PBMC stimulated with Con A released resistin, while adipocytes did not. CONCLUSION: Neither serum nor SF resistin were altered in dogs with OA secondary to CrCL insufficiency. In addition, resistin was not correlated with canine body fat and did not appear to function as adipocytokine in the dog. CLINICAL SIGNIFICANCE: Resistin may not be involved in the pathogenesis of OA. However, resistin may be important in inflammation because it is released from inflammatory cells.

Positive effects of platelet-rich plasma (PRP) and a Sanguisorba officinalis polysaccharide on the proliferation and differentiation of anterior cruciate ligament (ACL) fibroblasts in vitro.

Context: Sanguisorba officinalis L. (Rosaceae), a famous traditional Chinese medicine. It was recently reported that its polysaccharide could facilitate collagen production.Objectives: We investigated the mechanism by which S. officinalis polysaccharide (SOWPa) and/or platelet-rich plasma (PRP) promote regenerative potential of anterior cruciate ligament (ACL) in vitro.Materials and methods: ACL fibroblasts were treated with SOWPa (25 and 100 mg/kg), PRP, PRP + SOWPa (25 and 100 mg/kg) or vehicle alone for 24, 48, or 72 h. Cell viability, migration ability and apoptosis were evaluated by MTT, transwell and flow cytometry, respectively. Western blot analysis was performed to assess associated protein expression.Results: PRP, SOWPa (100 mg/kg) or PRP + SOWPa (100 mg/kg) treatment for 72 h significantly improved the cell viability of ACL fibroblasts from 100 ± 7.5% (control) to 156.85 ± 12.82%, 188.08 ± 15.92%, and 223.67 ± 18.82%, respectively, which was evidenced by individual decreased apoptosis rate from 31.26 ± 2.35% (control) to 20.80 ± 1.89%, 18.01 ± 1.55% and 9.33 ± 0.78%. Furthermore, the motility of ACL fibroblasts was significantly improved with increased migrated cell number per field from 5 for control to 26 for PRP, 36 for SOWPa and 44 for PRP + SOWPa, respectively. Moreover, the protein expression of differentiation markers (RUNX2, ALP, BMP2 and Col I) and TLR-4 and phosphorylated p65 (p-p65) was inhibited by the above treatment.Discussion and conclusions: Data suggested that the addition of SOWPa to PRP increased the regenerative ability of ACL fibroblasts by blocking the TLR-4/NF-κB pathway.

Proteomic analysis of synovial fluid identifies periostin as a biomarker for anterior cruciate ligament injury.

OBJECTIVE: Emerging evidence suggests that injury to the anterior cruciate ligament (ACL) typically initiates biological changes that contribute to the development of osteoarthritis (OA). The molecular biomarkers or mediators of these biological events remain unknown. The goal of this exploratory study was to identify novel synovial fluid biomarkers associated with early biological changes following ACL injury distinct from findings in end-stage OA. METHODS: Synovial fluid was aspirated from patients with acute (≤30 days) and subacute (31-90 days) ACL tears and from patients with advanced OA and probed via tandem mass spectrometry for biomarkers to distinguish OA from ACL injury. Periostin (POSTN) was identified as a potential candidate. Further analyses of POSTN were performed in synovial fluid, OA cartilage, torn ACL remnants, and cultured cells and media by Western blot, PCR, immunostaining and ELISA. RESULTS: Synovial fluid analysis revealed that POSTN exhibited higher expression in subacute ACL injury than OA. POSTN expression was relatively low in cartilage/chondrocytes suggesting it is also produced by other intra-articular tissues. Conversely, high and time-dependent expression of POSTN in ACL tear remnants and isolated cells was consistent with the synovial fluid results. CONCLUSIONS: Elevated POSTN may provide a synovial fluid biomarker of subacute ACL injury setting separate from OA. Increased expression of POSTN in ACL suggests that the injured ACL may play a pivotal role in POSTN production, which is sensitive to time from injury. Previous studies have shown potential catabolic effects of POSTN, raising the possibility that POSTN contributes to the initiation of joint degeneration and may offer a window of opportunity to intervene in the early stages of post-traumatic OA.

Short link N acts as a disease modifying osteoarthritis drug.

Osteoarthritis (OA) is a degenerative joint disease characterised by a progressive degradation of articular cartilage and underlaying bone and is associated with pain and disability. Currently, there is no medical treatment to reverse or even retard OA. Based on our previous reports, where we establish the repair potential of short Link N (sLN) in the intervertebral disc, a cartilage-like tissue, we hypothesise that sLN may hold similar promises in the repair of articular cartilage. This study aimed to determine if sLN, could prevent OA disease progression. Skeletally mature New Zealand white rabbits underwent unilateral anterior cruciate ligament transection (ACLT) of their left femorotibial joints to induce joint degeneration typical of OA. Beginning 3 weeks post-operatively, and every three weeks thereafter for 12 weeks, either saline (1 mL) or sLN (100 µg in 1 mL saline) was injected intraarticularly into the operated knee. Six additional rabbits underwent sham surgery but without ACLT or post-operative injections. The effects on gross joint morphology and cartilage histologic changes were evaluated. In the Saline group, prominent erosion of articular cartilage occurred in both femoral condyle compartments and the lateral compartment of the tibial plateau while, sLN treatment reduced the severity of the cartilage damage in these compartments of the knee showing erosion. Furthermore, statistically significant differences were detected between the joint OA score of the saline and sLN treated groups (p = 0.0118). Therefore, periodic intraarticular injection of sLN is a promising nonsurgical treatment for preventing or retarding OA progression, by reducing cartilage degradation.

Viscoelastic Behavior of Embroidered Scaffolds for ACL Tissue Engineering Made of PLA and P(LA-CL) After In Vitro Degradation.

A rupture of the anterior cruciate ligament (ACL) is the most common knee ligament injury. Current applied reconstruction methods have limitations in terms of graft availability and mechanical properties. A new approach could be the use of a tissue engineering construct that temporarily reflects the mechanical properties of native ligament tissues and acts as a carrier structure for cell seeding. In this study, embroidered scaffolds composed of polylactic acid (PLA) and poly(lactic-co-ε-caprolactone) (P(LA-CL)) threads were tested mechanically for their viscoelastic behavior under in vitro degradation. The relaxation behavior of both scaffold types (moco: mono-component scaffold made of PLA threads, bico: bi-component scaffold made of PLA and P(LA-CL) threads) was comparable to native lapine ACL. Most of the lapine ACL cells survived 32 days of cell culture and grew along the fibers. Cell vitality was comparable for moco and bico scaffolds. Lapine ACL cells were able to adhere to the polymer surfaces and spread along the threads throughout the scaffold. The mechanical behavior of degrading matrices with and without cells showed no significant differences. These results demonstrate the potential of embroidered scaffolds as an ACL tissue engineering approach.

Sustained intra-cartilage delivery of low dose dexamethasone using a cationic carrier for treatment of post traumatic osteoarthritis.

Disease-modifying osteoarthritis drugs (DMOADs) should reach their intra-tissue target sites at optimal doses for clinical efficacy. The dense, negatively charged matrix of cartilage poses a major hindrance to the transport of potential therapeutics. In this work, electrostatic interactions were utilised to overcome this challenge and enable higher uptake, full-thickness penetration and enhanced retention of dexamethasone (Dex) inside rabbit cartilage. This was accomplished by using the positively charged glycoprotein avidin as nanocarrier, conjugated to Dex by releasable linkers. Therapeutic effects of a single intra-articular injection of low dose avidin-Dex (0.5 mg Dex) were evaluated in rabbits 3 weeks after anterior cruciate ligament transection (ACLT). Immunostaining confirmed that avidin penetrated the full cartilage thickness and was retained for at least 3 weeks. Avidin-Dex suppressed injury-induced joint swelling and catabolic gene expression to a greater extent than free Dex. It also significantly improved the histological score of cell infiltration and morphogenesis within the periarticular synovium. Micro-computed tomography confirmed the reduced incidence and volume of osteophytes following avidin-Dex treatment. However, neither treatment restored the loss of cartilage stiffness following ACLT, suggesting the need for a combinational therapy with a pro-anabolic factor for enhancing matrix biosynthesis. The avidin dose used caused significant glycosaminoglycan (GAG) loss, suggesting the use of higher Dex : avidin ratios in future formulations, such that the delivered avidin dose could be much less than that shown to affect GAGs. This charge-based delivery system converted cartilage into a drug depot that could also be employed for delivery to nearby synovium, menisci and ligaments, enabling clinical translation of a variety of DMOADs.

Reconstruction of cranial cruciate ligament in rabbits using polyester implants saturated with PRP, antlerogenic stem cells MIC-1 and their homogenate.

AIM OF THE STUDY: The attempt to limit the negative effects of polyester implants on the articular cavity by using preparations containing growth factors. MATERIALS AND METHODS: Polyester implants used for the reconstruction of a rabbit's cranial cruciate ligament (CCL) were saturated with autogenic platelet-rich plasma (PRP), antlerogenic stem cells MIC-1 and their homogenate prior to the surgery. Six months after CCL reconstruction, morphological, and biochemical blood tests were carried out, including proteinogram and acute phase proteins. The knee joints were also examined macro- and microscopically. RESULTS: The results, compared to the control group, showed a favorable effect of the PRP and homogenate of antlerogenic cells on limiting the inflammation caused by the presence of polyester implant in the knee joint. The addition of growth factors caused covering the implant faster with the recipient's connective tissue, thus contributing to reducing the inflammatory reaction of the articular capsule to the presence of polyester. At the same time, no enhanced local or general reaction of the rabbit organism was observed to the presence of xenogenic antlerogenic stem cells MIC-1 homogenate which, like the PRP, may provide an easily available source of growth factors, increasingly often used in regenerative medicine. CONCLUSIONS: Applying antlerogenic stem cells, their homogenate or PRP increases the volume of connective tissue that surrounds and intertwines polyester CCL implant, separating it from synovial cavity environment.

Mechanical stretch and hypoxia inducible factor-1 alpha affect the vascular endothelial growth factor and the connective tissue growth factor in cultured ACL fibroblasts.

PURPOSES: The adult human anterior cruciate ligament (ACL) has poor functional healing response. Hypoxia plays an important role in regulating the microenvironment of the joint cavity after ACL injury, however, its role in mechanical injury is yet to be examined fully in ACL fibroblasts. In this study, we used CoCl2 to induce Hypoxia-inducible factor-1α (HIF-1α) in our experimental model to study its affect on matrix metalloproteinase-2 (MMP-2), vascular endothelial growth factor (VEGF), and connective tissue growth factor (CTGF) expression in ACL fibroblasts after mechanical stretch. MATERIALS AND METHODS: Cell treatments were performed in the stretch chamber in all experimental groups. Quantitative real-time PCR was used to check mRNA expression levels of MMP-2, CTGF, VEGF, and HIF-1α. Western blot was used to detect the HIF-1α production. Enzyme-Linked immunosorbent assay was performed to check the VEGF and CTGF protein contents in supernatant. MMP-2 activity was assayed by gelatin zymography. RESULTS: The real-time PCR results show that mechanical stretch or CoCl2 treatment increases the expression of MMP-2, VEGF, CTGF, and HIF-1α; however, the combined effects of mechanical stretch and CoCl2-induced HIF-1α increased MMP-2 production but decreased the VEGF and CTGF expression, compared to the CoCl2 treatment group alone. Western blot analysis and ELISA also confirmed these results. CONCLUSIONS: Our results demonstrated that mechanical stretch and CoCl2-induced HIF-1α together increased the level of MMP-2 and decreased the levels of VEGF and CTGF in cultured ACL fibroblasts. The differential expression and production of HIF-1α, VEGF, MMP-2, and CTGF might help to explain the poor healing ability of ACL.

[Effect of intermittent negative pressure on matrix metalloproteinase 9 and transforming growth factor ß of tendon-bone interface and joint fluid after reconstruction of anterior cruciate ligament in rabbits].

Objective: To study the effect of intermittent negative pressure on matrix metalloproteinase 9 (MMP)-9 and transforming growth factor ß of tendon-bone interface and joint fluid after reconstruction of anterior cruciate ligament in rabbits. Methods: A total of twenty-four New Zealand white rabbits were randomly selected hind leg of negative group, contralateral hind leg as control.Reconstruction of the anterior cruciate ligament was done by autogenous semitendinosus of rabbit.Joint of the negative pressure side placed drainage tube connecting the micro-negative pressure aspirator, and maintained an intermittent, low-intensity negative pressure.Control side placed ordinary drainage tube.Drainage tube of both sides was pulled out at the same time after 5 days.After 6 weeks, joint fluid and femur-ligament-tibia complex were obtained for study of expression of MMP-9 and TGF-ß in joint fluid and tendon-bone interface. Result: Twenty-three rabbits were included in the study because of one rabbit joint infections.Detection of joint fluid showed that MMP-9 content is significantly lower in negative group than that in the control group, and the difference is statistically significant [(8.9±1.3) pg/L vs (12.3±1.8) pg/L (P=0.002)]. TGF-ß content is significantly higher in negative group in joint fluid than that in the control group, and the difference is statistically significant [(19.0±2.2) pg/L vs (15.2±1.4) pg/L (P=0.000)]. Study of immunohistochemistry in tendon-bone interface found that expression of MMP-9 is lower in negative pressure group than that in the control group, and the difference is statistically significant (P=0.000). TGF-ß expression is significantly higher in negative group in tendon-bone interface than that in the control group, and the difference is statistically significant (P=0.000). Conclusion: Intermittent negative pressure may reduce content of MMP-9 in joint fluid and expression of MMP-9 in tendon-bone interface, increase content of TGF-ß in joint fluid and expression of TGF-ß in tendon-bone interface after reconstruction of anterior cruciate ligament in rabbits.

Proteomic differences between native and tissue-engineered tendon and ligament.

Tendons and ligaments (T/Ls) play key roles in the musculoskeletal system, but they are susceptible to traumatic or age-related rupture, leading to severe morbidity as well as increased susceptibility to degenerative joint diseases such as osteoarthritis. Tissue engineering represents an attractive therapeutic approach to treating T/L injury but it is hampered by our poor understanding of the defining characteristics of the two tissues. The present study aimed to determine differences in the proteomic profile between native T/Ls and tissue engineered (TE) T/L constructs. The canine long digital extensor tendon and anterior cruciate ligament were analyzed along with 3D TE fibrin-based constructs created from their cells. Native tendon and ligament differed in their content of key structural proteins, with the ligament being more abundant in fibrocartilaginous proteins. 3D T/L TE constructs contained less extracellular matrix (ECM) proteins and had a greater proportion of cellular-associated proteins than native tissue, corresponding to their low collagen and high DNA content. Constructs were able to recapitulate native T/L tissue characteristics particularly with regard to ECM proteins. However, 3D T/L TE constructs had similar ECM and cellular protein compositions indicating that cell source may not be an important factor for T/L tissue engineering.