CXCR4 antagonist delivery on decellularized skin scaffold facilitates impaired wound healing in diabetic mice by increasing expression of SDF‐1 and enhancing migration of CXCR4‐positive cells

C‐X‐C chemokine receptor type 4 (CXCR4) is an alpha‐chemokine receptor specific for stromal cell‐derived factor 1 (SDF‐1 also called CXCL12). The antagonist of CXCR4 can mobilize CD34+ cells and hematopoietic stem cells from bone marrow within several hours, and it has an efficacy on diabetes ulcer through acting on the SDF‐1/CXCR4 axis. In this study, we investigated for the first time whether the antagonist of CXCR4 (Plerixafor/AMD3100) delivered on acellular dermal matrix (ADM) may accelerate diabetes‐impaired wound healing. ADM scaffolds were fabricated from nondiabetic mouse skin through decellularization processing and incorporated with AMD3100 to construct ADM‐AMD3100 scaffold. Full‐thickness cutaneous wound in streptozotocin (STZ)‐induced diabetic mice were treated with ADM, AMD3100, or ADM‐AMD3100. 21 days after treatment, wound closure in ADM‐AMD3100‐treated mice was more complete than ADM group and AMD3100 group, and it was accompanied by thicker collagen formation. Correspondingly, diabetic mice treated with ADM‐AMD3100 demonstrated prominent neovascularization (higher capillary density and vascular smooth muscle actin), which were accompanied by up‐regulated mRNA levels of SDF‐1 and enhanced migration of CXCR4 in the granulation tissue. Our results demonstrate that ADM scaffold provide perfect niche for loading AMD3100 and ADM‐AMD3100 is a promising method for diabetic wound healing mainly by increasing expression of SDF‐1 and enhancing migration of CXCR4‐positive cells.     

Long‐term administration of AMD3100, an antagonist of SDF‐1/CXCR4 signaling,alters fracture repair

Fracture healing involves rapid stem and progenitor cell migration, homing, and differentiation. SDF‐1 (CXCL12) is considered a master regulator of CXCR4‐positive stem and progenitor cell trafficking to sites of ischemic (hypoxic) injury and regulates their subsequent differentiation into mature reparative cells. In this study, we investigated the role of SDF‐1/CXCR4 signaling in fracture healing where vascular disruption results in hypoxia and SDF‐1 expression. Mice were injected with AMD3100, a CXCR4 antagonist, or vehicle twice daily until euthanasia with the intent to impair stem cell homing to the fracture site and/or their differentiation. Fracture healing was evaluated using micro‐computed tomography, histology, quantitative PCR, and mechanical testing. AMD3100 administration resulted in a significantly reduced hyaline cartilage volume (day 14), callus volume (day 42) and mineralized bone volume (day 42) and reduced expression of genes associated with endochondral ossification including collagen Type 1 alpha 1, collagen Type 2 alpha 1, vascular endothelial growth factor, Annexin A5, nitric oxide synthase 2, and mechanistic target of rapamycin. Our data suggest that the SDF‐1/CXCR4 signaling plays a central role in bone healing possibly by regulating the recruitment and/or differentiation of stem and progenitor cells. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1853–1859, 2012     

Characterization of Post-Traumatic Osteoarthritis in Rats Following Anterior Cruciate Ligament Rupture by Non-Invasive Knee Injury (NIKI)

Small animal models are essential for studying anterior cruciate ligament (ACL) injury, one of the leading risk factors for post-traumatic osteoarthritis (PTOA). Non-surgical models of ACL rupture have recently surged as a new tool to study PTOA, as they circumvent the confounding effects of surgical disruption of the joint. These models primarily have been explored in mice and rabbits, but are relatively understudied in rats. The purpose of this work was to establish a non-invasive, mechanical overload model of ACL rupture in the rat and to study the disease pathogenesis following the injury. ACL rupture was induced via non-invasive tibial compression in Lewis rats. Disease state was characterized for 4 months after ACL rupture via histology, computed tomography, and biomarker capture from the synovial fluid. The non-invasive knee injury (NIKI) model created consistent ACL ruptures without direct damage to other tissues and resulted in conventional OA pathology. NIKI knees exhibited structural changes as early as 4 weeks post-injury, including regional structural changes to cartilage, chondrocyte and cartilage disorganization, changes to the bone architecture, synovial hyperplasia, and the increased presence of biomarkers of cartilage fragmentation and pro-inflammatory cytokines. These results suggest that this model can be a valuable tool to study PTOA. By establishing the fundamental pathogenesis of this injury, additional opportunities are created to evaluate unique contributing factors and potential therapeutic interventions for this disease. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:356-367, 2020     

Suppression of murine osteoarthritis by 4-methylumbelliferone

Using in vitro models, we previously reported that 4-methylumbelliferone (4-MU) blocked many of the pro-catabolic features of activated chondrocytes. 4-MU also blocked safranin O loss from human cartilage explants exposed to interleukin 1β (IL1β) in vitro. However, the mechanism for this chondroprotective effect was independent of the action of 4-MU as a hyaluronan (HA) inhibitor. Interestingly, overexpression of HA synthase 2 (HAS2) also blocked the same pro-catabolic features of activated chondrocytes as 4-MU via a mechanism independent of extracellular HA accumulation. Data suggest that altering UDP-sugars may be behind these changes in chondrocyte metabolism. However, all of our previous experiments with 4-MU or HAS2 overexpression were performed in vitro. The purpose of this study was to confirm whether 4-MU was effective at limiting the effects of osteoarthritis (OA) on articular cartilage in vivo. The progression of OA was evaluated after destabilization of the medial meniscus (DMM) surgery on C57BL/6 mice in the presence or absence of 4-MU-containing chow. Mice fed 4-MU after DMM surgery exhibited significant suppression of OA starting from an early stage in vivo. Mice fed 4-MU exhibited lower OARSI scores after DMM; reduced osteophyte formation and reduced MMP3 and MMP13 immunostaining. 4-MU also exerted pronounced chondroprotective effects on murine joint cartilage exposed to IL1β in vitro and, blocked IL1β-enhanced lactate production in cartilage explants. Therefore, 4-MU is effective at significantly reducing the loss of proteoglycan and reducing MMP production both in vitro and in vivo as well as cartilage damage and osteophyte formation in vivo after DMM. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res. 38:1122-1131, 2020     

CXCR4 antagonism attenuates load‐induced periosteal bone formation in mice

Mechanical loading is a key anabolic regulator of bone mass. Stromal cell‐derived factor‐1 (SDF‐1) is a stem cell homing factor that is important in hematopoiesis, angiogenesis, and fracture healing, though its involvement in skeletal mechanoadaptation is virtually unknown. The objective of this study was to characterize skeletal expression patterns of SDF‐1 and CXCR4, the receptor for SDF‐1, and to determine the role of SDF‐1 signaling in load‐induced periosteal bone formation. Sixteen‐week‐old C57BL/6 mice were treated with PBS or AMD3100, an antagonist against CXCR4, and exposed to in vivo ulnar loading (2.8 N peak‐to‐peak, 2 Hz, 120 cycles). SDF‐1 was expressed in cortical and trabecular osteocytes and marrow cells, and CXCR4 was primarily expressed in marrow cells. SDF‐1 and CXCR4 expression was enhanced in response to mechanical stimulation. The CXCR4 receptor antagonist AMD3100 significantly attenuated load‐induced bone formation and led to smaller adaptive changes in cortical geometric properties as determined by histomorphometric analysis. Our data suggest that SDF‐1/CXCR4 signaling plays a critical role in skeletal mechanoadaptation, and may represent a unique therapeutic target for prevention and treatment of age‐related and disuse bone loss. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:1828–1838, 2013     

Early inhibition of subchondral bone remodeling slows load-induced posttraumatic osteoarthritis development in mice

Posttraumatic osteoarthritis (PTOA) is associated with abnormal and increased subchondral bone remodeling. Inhibiting altered remodeling immediately following joint damage can slow PTOA progression. Clinically, however, inhibiting remodeling when significant joint damage is already present has minimal effects in slowing further disease progression. We sought to determine the treatment window following PTOA initiation in which inhibiting remodeling can attenuate progression of joint damage. We hypothesized that the most effective treatment would be to inhibit remodeling immediately after PTOA initiation. We used an animal model in which a single bout of mechanical loading was applied to the left tibia of 26-week-old male C57Bl/6 mice at a peak load of 9 N to initiate load-induced PTOA development. Following loading, we inhibited bone remodeling using daily alendronate (ALN) treatment administered either immediately or with 1 or 2 weeks' delay up to 3 or 6 weeks post-loading. A vehicle (VEH) treatment group controlled for daily injections. Cartilage and subchondral bone morphology and osteophyte development were analyzed and compared among treatment groups. Inhibiting remodeling using ALN immediately after load-induced PTOA initiation reduced cartilage degeneration, slowed osteophyte formation, and preserved subchondral bone volume compared to VEH treatment. Delaying the inhibition of bone remodeling at 1 or 2 weeks similarly attenuated cartilage degeneration at 6 weeks, but did not slow the development of osteoarthritis (OA)-related changes in the subchondral bone, including osteophyte formation and subchondral bone erosions. Immediate inhibition of subchondral bone remodeling was most effective in slowing PTOA progression across the entire joint, indicating that abnormal bone remodeling within the first week following PTOA initiation played a critical role in subsequent cartilage damage, subchondral bone changes, and overall joint degeneration. These results highlight the potential of anti-resorptive drugs as preemptive therapies for limiting PTOA development after joint injury, rather than as disease-modifying therapies after joint damage is established. © 2021 American Society for Bone and Mineral Research (ASBMR).     

Transient expression of the diseased phenotype of osteoarthritic chondrocytes in engineered cartilage

Due to the degradation of osteoarthritic (OA) cartilage in post‐traumatic OA (PTOA), these tissues are challenging to study and manipulate in vitro. In this study, chondrocytes isolated from either PTOA (meniscal‐release (MR) model) or normal (contralateral limb) cartilage of canine knee joints were used to form micropellets to assess the maintenance of the OA chondrocyte phenotype in vitro. Media samples from the micropellet cultures were used to measure matrix metalloproteinase (MMP), chemokine, and cytokine concentrations. Significant differences in matrix synthesis were observed as a function of disease with OA chondrocytes generally synthesizing more extracellular matrix with increasing time in culture. No donor dependent differences were detected. Luminex multiplex analysis of pellet culture media showed disease and time‐dependent differences in interleukin (IL)‐8, keratinocyte chemoattractant (KC)‐like protein, MMP‐1, MMP‐2, and MMP‐3, which are differentially expressed in OA. This memory of their diseased phenotype persists for the first 2 weeks of culture. These results demonstrate the potential to use chondrocytes from an animal model of OA to study phenotype alterations during the progression and treatment of OA. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:829–836, 2017.

     

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

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

SOST/Sclerostin Improves Posttraumatic Osteoarthritis and Inhibits MMP2/3 Expression After Injury

Patients with anterior cruciate ligament (ACL) rupture are two times as likely to develop posttraumatic osteoarthritis (PTOA). Annually, there are ～900,000 knee injuries in the United States, which account for ～12% of all osteoarthritis (OA) cases. PTOA leads to reduced physical activity, deconditioning of the musculoskeletal system, and in severe cases requires joint replacement to restore function. Therefore, treatments that would prevent cartilage degradation post‐injury would provide attractive alternatives to surgery. Sclerostin (Sost), a Wnt antagonist and a potent negative regulator of bone formation, has recently been implicated in regulating chondrocyte function in OA. To determine whether elevated levels of Sost play a protective role in PTOA, we examined the progression of OA using a noninvasive tibial compression overload model in SOST transgenic (SOST^TG) and knockout (Sost^‐/‐) mice. Here we report that SOST^TG mice develop moderate OA and display significantly less advanced PTOA phenotype at 16 weeks post‐injury compared with wild‐type (WT) controls and Sost^‐/‐. In addition, SOST^TG built ～50% and ～65% less osteophyte volume than WT and Sost^‐/‐, respectively. Quantification of metalloproteinase (MMP) activity showed that SOST^TG had ～2‐fold less MMP activation than WT or Sost^‐/‐, and this was supported by a significant reduction in MMP2/3 protein levels, suggesting that elevated levels of SOST inhibit the activity of proteolytic enzymes known to degrade articular cartilage matrix. Furthermore, intra‐articular administration of recombinant Sost protein, immediately post‐injury, also significantly decreased MMP activity levels relative to PBS‐treated controls, and Sost activation in response to injury was TNFα and NF‐κB dependent. These results provide in vivo evidence that sclerostin functions as a protective molecule immediately after joint injury to prevent cartilage degradation. © 2018 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.     

AMD3100体外阻断基质细胞衍生因子1/趋化因子受体4信号通路对人关节软骨细胞分泌基质金属蛋白酶3、9、13水平的影响

目的探讨趋化因子受体4(chemokine receptor 4,CXCR4)特异性拮抗剂AMD3100体外阻断基质细胞衍生因子1(stromal cell derived factor 1,SDF-1)/CXCR4信号通路对人关节软骨细胞分泌基质金属蛋白酶(matrixmetalloproteinase,MMP)3、9、13水平的影响,明确AMD3100的作用机制。方法取12例骨关节炎(osteoarthritis,OA)患者144块软骨组织(OA软骨组)和12例创伤性截肢患者144块正常软骨组织(正常软骨组)(Mankin评分均为0或1),根据添加培养液不同,每组再分为A、B、C 3个亚组。A亚组含1 000 nmol/L AMD3100及100 ng/mL SDF-1的DMEM液,B亚组含1 000 nmol/L MAB310及100 ng/mL SDF-1的DMEM液,C亚组含100 ng/mL SDF-1的DMEM液。体外培养2、4 d后,ELISA法测定培养液内MMP-3、9、13含量,RT-PCR检测软骨组织中MMP-3、9、13 mRNA表达。结果 ELISA法及RT-PCR检测示:同组相同时间点A亚组MMP-3、9、13含量及mRNA表达均显著低于B、C亚组(P<0.05)。同一时间点相同亚组OA软骨组MMP-3、9、13含量及mRNA表达均显著高于正常软骨组(P<0.05)。结论 SDF-1通过SDF-1/CXCR4信号通路可诱导人关节软骨中MMP-3、9、13的表达和释放;AMD3100可阻断SDF-1/CXCR4信号通路,使软骨细胞MMP-3、9、13 mRNA表达水平及分泌量降低,但AMD3100不能使退变的OA软骨分泌MMP-3、9、13恢复至正常软骨水平。     

A single blunt impact on cartilage promotes fibronectin fragmentation and upregulates cartilage degrading stromelysin‐1/matrix metalloproteinase‐3 in a bovine ex vivo model

Post‐traumatic osteoarthritis (PTOA) is characterized by progressive cartilage degeneration in injured joints. Since fibronectin‐fragments (Fn‐fs) degrade cartilage mainly through up‐regulating matrix metalloproteinases (MMPs) and pro‐inflammatory cytokines, we hypothesized that Fn‐fs play a key role in PTOA by promoting chondrolysis in and around injured cartilage. To test this hypothesis, we profiled the catabolic events focusing on fibronectin fragmentation and proteinase expression in bovine osteochondral explants following a single blunt impact on cartilage with a drop tower device which created partial‐thickness tissue damage. Injured and control explants were cultured for up to 14 days. The presence of Fn‐fs, MMPs (‐1, ‐3, ‐13), ADAMTS‐5 in culture media and in cartilage was determined with immunoblotting. The daily proteoglycan (PG) depletion of cartilage matrix was assessed with DMMB assay. The effect of explant‐conditioned media on chondrocytes was also examined with immunoblotting. Impacted cartilage released significantly higher amount of native Fn, three chondrolytic Fn‐fs and PG than non‐impacted controls did. Those increases coincided with up‐regulation of MMP‐3 both in culture media and in impacted cartilage. These findings support our hypothesis that PTOA may be propelled by Fn‐fs which act as catabolic mediators through up‐regulating cartilage‐damaging proteinases. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:811–818, 2014.     

Inflammatory and degenerative phases resulting from anterior cruciate rupture in a non‐invasive murine model of post‐traumatic osteoarthritis

Joint injury is the predominant risk factor for post‐traumatic osteoarthritis development (PTOA). Several non‐invasive mouse models mimicking human PTOA investigate molecular mechanisms of disease development; none have characterized the inflammatory response to this acute traumatic injury. Our aim was to characterize the early inflammatory phase and later degenerative component in our in vivo non‐invasive murine model of PTOA induced by anterior cruciate ligament (ACL) rupture. Right knees of 12‐week‐old C57Bl6 mice were placed in flexion at a 30° offset position and subjected to a single compressive load (12N, 1.4 mm/s) to induce ACL rupture with no obvious damage to surrounding tissues. Tissue was harvested 4 h post‐injury and on days 3, 14, and 21; contralateral left knees served as controls. Histological, immunohistochemical, and gene analyzes were performed to evaluate inflammatory and degenerative changes. Immunohistochemistry revealed time‐dependent expression of mature (F4/80 positive) and inflammatory (CD11b positive) macrophage populations within the sub‐synovial infiltrate, developing osteophytes, and inflammation surrounding the ACL in response to injury. Up‐regulation of genes encoding acute pro‐inflammatory markers, inducible nitric oxide synthase, interleukin‐6 and interleukin‐17, and the matrix degrading enzymes, ADAMTS‐4 and MMP3 was detected in femoral cartilage, concomitant with extensive cartilage damage and bone remodelling over 21‐days post‐injury. Our non‐invasive model describes pathologically distinct phases of the disease, increasing our understanding of inflammatory episodes, the tissues/cells producing inflammatory mediators and the early molecular changes in the joint, thereby defining the early phenotype of PTOA. This knowledge will guide appropriate interventions to delay or arrest disease progression following joint injury. © 2018 The Authors. Journal of Orthopaedic Research® Published by Wiley Periodicals, Inc. on behalf of the Orthopaedic Research Society. J Orthop Res 36:2118–2127, 2018.     

Utilization of longitudinal ultrasound to quantify joint soft-tissue changes in a mouse model of posttraumatic osteoarthritis

To assess the utility of longitudinal ultrasound(US) to quantify volumetric changes in joint soft tissues during the progression of posttraumatic osteoarthritis(PTOA) in mice, and validate the US results with histological findings. A longitudinal cohort of 3-month-old wild-type C57 BL/6 male mice received the Hulth-Telhag surgical procedure on right knee to induce PTOA, and sham surgery on their left knee as control. US scans were performed on both knees before, 2, 4, 6, and 8 weeks post-surgery. Joint space volume and Power-Doppler(PD) volume were obtained from US images via Amira software. A parallel cross-sectional cohort of mice was killed at each US time point, and knee joints were subjected to histological analysis to obtain synovial soft-tissue area and OARSI scores. The correlation between US joint space volume and histological synovial soft-tissue area or OARSI score was assessed via linear regression analysis. US images indicated increased joint space volume in PTOA joints over time, which was associated with synovial inflammation and cartilage damage by histology. These changes started from 2 weeks post-surgery and gradually became more severe. No change was detected in sham joints. Increased joint space volume was significantly correlated with increased synovial soft-tissue area and the OARSI score(P o 0.001). PD signal was detected in the joint space of PTOA joints at 6 weeks post-surgery, which was consistent with the location of blood vessels that stained positively for CD31 and alpha-smooth muscle actin in the synovium.This study indicates that US is a cost-effective longitudinal outcome measure of volumetric and vascular changes in joint soft tissues during PTOA progression in mice, which positively correlates with synovial inflammation and cartilage damage.     

SDF-1/CXCR4信号轴调控骨髓间充质干细胞向哮喘小鼠肺组织的迁移

目的：探讨基质细胞衍生因子-1（SDF-1）/CXCR4轴在外源性骨髓间充质干细胞（MSC）向哮喘模型小鼠肺组织迁移的作用.方法：无菌条件下取GFP转基因小鼠骨髓MSC,体外扩增,鉴定.采用Transwell培养系统,观察0、50、100、150和200 ng/ml SDF-1和CXCR4阻断剂AMD3100对MSC体外定向迁移的影响.取60只雌性BALB/c小鼠,随机分为6组（n=10）：PBS非哮喘组、MSC非哮喘组、PBS哮喘组、MSC哮喘组、SDF-1处理哮喘组、AMD3100处理哮喘组.哮喘组采用哮喘致敏液0.2 ml（含100 μg卵白蛋白）致敏,并使用卵白蛋白雾化吸入激发哮喘.非哮喘组在致敏和激发时均予以PBS处理.MSC处理组于哮喘激发前移植外源性MSC.SDF-1处理哮喘组在MSC移植前气管内注入SDF-1,AMD3100处理哮喘组注入AMD3100预先孵育的MSC.PBS哮喘组注射等量的PBS液.采用Westernablot和RT-PCR方法检测肺组织中SDF-1的表达水平,通过荧光显微镜观察表达GFP的外源性MSC在哮喘小鼠肺组织中的分布情况,比较SDF-1和CXCR4阻断剂AMD3100干预对MSC向肺组织迁移的影响.结果：Transwell实验显示MSC的迁移水平与SDF-1（0~150军ng/ml）成浓度依赖性.与正常小鼠比较,哮喘小鼠肺组织SDF-1表达增强.与MSC非哮喘组比较,MSC哮喘组小鼠肺部有更多MSC聚集.在哮喘肺组织中增加外源性SDF-1能够促进MSC向肺组织迁移.通过AMD3100阻断MSC的CXCR4可以明显减少MSC向肺组织的迁移水平.结论：SDF-1/CXCR4轴参与了MSC迁移到哮喘小鼠肺组织的过程.     

In Vivo Dual Fluorescence Imaging to Detect Joint Destruction

Diagnosis of cartilage damage in early stages of arthritis is vital to impede the progression of disease. In this regard, considerable progress has been made in near‐infrared fluorescence (NIRF) optical imaging technique. Arthritis can develop due to various mechanisms but one of the main contributors is the production of matrix metalloproteinases (MMPs), enzymes that can degrade components of the extracellular matrix. Especially, MMP‐1 and MMP‐13 have main roles in rheumatoid arthritis and osteoarthritis because they enhance collagen degradation in the process of arthritis. We present here a novel NIRF imaging strategy that can be used to determine the activity of MMPs and cartilage damage simultaneously by detection of exposed type II collagen in cartilage tissue. In this study, retro‐orbital injection of mixed fluorescent dyes, MMPSense 750 FAST (MMP750) dye and Alexa Fluor 680 conjugated monoclonal mouse antibody immune‐reactive to type II collagen, was administered in the arthritic mice. Both dyes were detected with different intensity according to degree of joint destruction in the animal. Thus, our dual fluorescence imaging method can be used to detect cartilage damage as well as MMP activity simultaneously in early stage arthritis.     

Cyclin‐Dependent Kinase Inhibitor‐1‐Deficient Mice are Susceptible to Osteoarthritis Associated with Enhanced Inflammation

Osteoarthritis (OA) is a multifactorial disease, and recent data suggested that cell cycle–related proteins play a role in OA pathology. Cyclin‐dependent kinase (CDK) inhibitor 1 (p21) regulates activation of other CDKs, and recently, we reported that p21 deficiency induced susceptibility to OA induced by destabilization of the medial meniscus (DMM) surgery through STAT3‐signaling activation. However, the mechanisms associated with why p21 deficiency led to susceptibility to OA by the STAT3 pathway remain unknown. Therefore, we focused on joint inflammation to determine the mechanisms associated with p21 function during in vitro and in vivo OA progression. p21‐knockout (p21^?/?) mice were used to develop an in vivo OA model, and C57BL/6 (p21^+/+) mice with the same background as the p21^?/? mice were used as controls. Morphogenic changes were measured using micro‐CT, IL‐1β serum levels were detected by ELISA, and histological or immunohistological analyses were performed. Our results indicated that p21‐deficient DMM‐model mice exhibited significant subchondral bone destruction and cartilage degradation compared with wild‐type mice. Immunohistochemistry results revealed p21^?/? mice susceptibility to OA changes accompanied by macrophage infiltration and enhanced MMP‐3 and MMP‐13 expression through IL‐1β‐induced NF‐κB signaling. p21^?/? mice also showed subchondral bone destruction according to micro‐CT analysis, and cathepsin K staining revealed increased numbers of osteoclasts. Furthermore, p21^?/? mice displayed increased serum IL‐1β levels, and isolated chondrocytes from p21^?/? mice indicated elevated MMP‐3 and MMP‐13 expression with phosphorylation of IκB kinase complex in response to IL‐1β stimulation, whereas treatment with a specific p‐IκB kinase inhibitor attenuated MMP‐3 and MMP‐13 expression. Our results indicated that p21‐deficient DMM mice were susceptible to alterations in OA phenotype, including enhanced osteoclast expression, macrophage infiltration, and MMP expression through IL‐1β‐induced NF‐κB signaling, suggesting that p21 regulation may constitute a possible therapeutic strategy for OA treatment. © 2017 American Society for Bone and Mineral Research.     

Mitoprotective therapy preserves chondrocyte viability and prevents cartilage degeneration in an ex vivo model of posttraumatic osteoarthritis

No disease‐modifying osteoarthritis (OA) drugs are available to prevent posttraumatic osteoarthritis (PTOA). Mitochondria (MT) mediate the pathogenesis of many degenerative diseases, and recent evidence indicates that MT dysfunction is a peracute (within minutes to hours) response of cartilage to mechanical injury. The goal of this study was to investigate cardiolipin‐targeted mitoprotection as a new strategy to prevent chondrocyte death and cartilage degeneration after injury. Cartilage was harvested from bovine knee joints and subjected to a single, rapid impact injury (24.0 ±1.4 MPa, 53.8 ± 5.3 GPa/s). Explants were then treated with a mitoprotective peptide, SS‐31 (1µM), immediately post‐impact, or at 1, 6, or 12 h after injury, and then cultured for up to 7 days. Chondrocyte viability and apoptosis were quantified in situ using confocal microscopy. Cell membrane damage (lactate dehydrogenase activity) and cartilage matrix degradation (glycosaminoglycan loss) were quantified in cartilage‐conditioned media. SS‐31 treatment at all time points after impact resulted in chondrocyte viability similar to that of un‐injured controls. This effect was sustained for up to a week in culture. Further, SS‐31 prevented impact‐induced chondrocyte apoptosis, cell membrane damage, and cartilage matrix degeneration. Clinical Significance: This study is the first investigation of cardiolipin‐targeted mitoprotective therapy in cartilage. These results suggest that even when treatment is delayed by up to 12 h after injury, mitoprotection may be a useful strategy in the prevention of PTOA. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2147–2156, 2018.

     

Single intra‐articular dexamethasone injection immediately post‐surgery in a rabbit model mitigates early inflammatory responses and post‐traumatic osteoarthritis‐like alterations

Despite surgical reconstruction of the anterior cruciate ligament, a significant number of patients will still develop post‐traumatic osteoarthritis (PTOA). Our objective was to determine if mitigating aspects of the acute phase of inflammation following a defined knee surgery with a single administration of a glucocorticoid could prevent the development of PTOA‐like changes within an established rabbit model of surgically induced PTOA. An early and late post‐surgical time‐point was investigated in this study (48 h and 9 weeks post‐surgery) in which the following groups were repeated (each n = 6, for a total of 24 rabbits per time‐point, and 48 rabbits used in the study): control (age/sex matched), sham (arthrotomy), drill injury (arthrotomy + two drill holes to a non‐cartilaginous area of the femoral notch), and drill injury + single intra‐articular (IA) injection of dexamethasone (DEX). At 48 h post‐surgery, DEX treatment significantly lowered the mRNA levels for a subset of pro‐inflammatory mediators, and significantly lowered the histological grade. Nine weeks post surgery, DEX treatment significantly lowered the histological scores (presented as effect size) for synovium (3.8), lateral femoral condyle (3.9), and lateral tibial cartilage (5.1) samples. Thus, DEX likely acts to prevent injury induced inflammation that could contribute to subsequent joint damage. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1826–1834, 2015.     

Effective knock down of matrix metalloproteinase‐13 by an intra‐articular injection of small interfering RNA (siRNA) in a murine surgically‐induced osteoarthritis model

This study investigated the effect of MMP‐13 gene knock down on cartilage degradation by injecting small interfering RNA (siRNA) into knee joints in a mouse model of osteoarthritis (OA). OA was induced in male C57BL/6 mice by destabilization of medial meniscus (DMM) surgery. Change of Mmp13 expression over time was determined by qPCR analysis from 3 days to 6 weeks after surgery. Mmp13 and control chemically modified siRNA were injected into the knee joint 1 week after surgery and expression levels were assessed in synovium by qPCR 48 h later. Cartilage degradation was histologically assessed 8 weeks after DMM surgery according to OARSI recommendations. Mmp13 expression levels were elevated 1 week after surgery and peaked at 77 fold at 2 weeks compared to expression at 3 days. A 55% decrease of Mmp13 levels in cartilage was observed 48 h after injection of Mmp13 siRNA (p = 0.05). Significant reduction in the histological score at 8 weeks after surgery was observed in the Mmp13 siRNA‐treated group compared to the control siRNA group (p < 0.001). Intra‐articular injection of Mmp13 siRNA at the early phase of OA development resulted in effective knock down of Mmp13 expression and delay in cartilage degradation in vivo. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1175–1180, 2014.     

Comparison of differential biomarkers of osteoarthritis with and without posttraumatic injury in the Hartley guinea pig model

The objective was to compare biomarkers of articular cartilage metabolism in synovial fluid from Hartley guinea pig knees, with and without anterior cruciate ligament transection (ACLT), to establish whether detectable differences in biomarker levels exist between primary and secondary osteoarthritis (OA). Synovial fluid lavages and knees were obtained from 3‐month (control group) and 12‐month (primary OA group) animals. Another group of animals (posttraumatic OA group) underwent unilateral ACLT at 3 months, and samples were obtained 9 months postsurgery. Synovial fluid concentrations of stromal cell‐derived‐factor (SDF‐1), collagen fragments (C2C), proteoglycan (GAG), lubricin, matrix metalloproteinase‐13 (MMP‐13), and Interleukin‐1 (IL‐1β) were evaluated. Cartilage damage was assessed via histology. The highest concentrations of C2C and SDF‐1 in synovial fluid were found in the posttraumatic OA group, moderate concentrations were found in the primary OA group, and low concentrations in the control group. GAG release in synovial fluid was similar to C2C and SDF‐1. The lubricin concentrations were significantly lower in ACLT joints than either the control or 12‐month primary OA groups, but not between the control and primary OA groups. Higher levels of MMP‐13 and IL‐1β were detected in the joints of the posttraumatic OA group as compared to the control or primary OA groups. Histology revealed greatest OA damage in the posttraumatic OA group, followed by moderate and minimal damage in primary OA and control groups, respectively. This study indicates that the biomarkers and progression of OA may differ in the Hartley guinea pig models with and without posttraumatic OA. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:900–906, 2010