Dudakovic, Amel

Arthrofibrosis is a common complication following total knee arthroplasty and is the result of a dysregulated immuno-inflammatory cascade, which culminates in excessive scar deposition by myofibroblasts within the knee joint. Pharmacotherapies for arthrofibrosis are limited, with treatment reliant on manipulation under anesthesia or lysis of adhesions with or without component revision. Bupivacaine (a local anesthetic) and meloxicam (a nonsteroidal anti-inflammatory drug) combination therapy may exert antifibrotic properties by limiting inflammatory cell recruitment and the subsequent release of profibrotic cytokines. The purpose of this study was to evaluate the effects of this combination therapy in a rabbit model of arthrofibrosis by evaluating live and postsacrifice knee biomechanics and gene expression of posterior knee capsule tissue. Forty New Zealand white rabbits were equally divided into two experimental groups and prospectively studied to assess knee passive extension angles (PEA), terminal posterior capsular stiffness, and the inflammatory milieu of the posterior knee joint capsule. Experimental limbs with and without combination therapy showed similar live PEAs and terminal posterior capsular stiffness, with no significant differences between the groups. Gene expression analysis, however, revealed significant reduction in inflammatory genes but not profibrotic genes. In summary, bupivacaine and meloxicam combination therapy did not exert antifibrotic effects in a rabbit model of arthrofibrosis but significantly reduced the expression of inflammatory markers in the local environment of the posterior capsule.

AimsTo characterize the effects of varying experimental parameters of transforming growth factor-beta 1 (TGF-β1) in primary knee fibroblasts as an in vitro model of arthrofibrosis.MethodsPrimary knee fibroblasts were isolated from one patient undergoing primary total knee arthroplasty (TKA) and one patient undergoing revision TKA for arthrofibrosis, respectively. Fibroblasts were stimulated with varying concentrations and durations of the pro-fibrotic cytokine TGF-β1. Picrosirius red staining (PSR) was conducted to assess collagen deposition, and real-time quantitative polymerase chain reaction (RT-qPCR) and western blotting were performed to determine cellular gene expression levels. A collagen gel contraction assay was used to assess TGF-β1-induced fibroblast contraction.ResultsIn our experiments, TGF-β1-mediated collagen deposition was consistent across a vast concentration range (0.5 to 40 ng/ml). While α-smooth muscle actin (ACTA2) protein levels and SMAD2 phosphorylation were induced by low concentrations (1 ng/ml), robust ACTA2 transcription required higher concentrations (5 ng/ml) of TGF-β1. Evaluation of TGF-β1-mediated cell and cytoskeletal contractility shows that in the measured fibroblasts, this process occurs within three hours following TGF-β1 administration. Of note, TGF-β1-differentiated myofibroblasts exhibited greater contractile properties than naïve fibroblasts in the presence of TGF-β1.ConclusionTaken together, this study elucidates key TGF-β1 experimental parameters that will inform the development and interpretation of in vitro models of arthrofibrosis.Cite this article: Bone Joint Res 2025;14(6):527–538.

Experimental analyses of knee joint contractures have traditionally utilized a 6-month rabbit model as the gold standard. However, this model is time-intensive and costly. The purpose of this study was to develop an abbreviated rabbit model of knee contractures and compare it to the well-established longer model. Twenty female New Zealand White rabbits were divided into two equal groups and prospectively studied to assess knee passive extension angles (PEA), contracture angles (CA), and terminal posterior capsular stiffness. Experimental knees were immobilized for either 4 weeks ( n = 10) with an 8-week remobilization period in the abbreviated model (i.e., 3 months) or for 8 weeks ( n = 10) with a 16-week remobilization period in the standard model (i.e., 6 months). PEAs were assessed at remobilization and several time points using differing vertical forces. At sacrifice, terminal biomechanical data were collected to assess posterior capsular stiffness. Analysis of PEAs in live animals at each torque value and time point demonstrated increased PEAs and decreased CAs in the 3-month abbreviated model as compared to the 6-month standard model. At sacrifice, biomechanical analysis demonstrated that the posterior capsules of the 3-month experimental limbs were significantly more stiff than the contralateral limb (2.4 vs. 0.05 Ncm/°, p < 0.0001), but significantly less stiff compared to the 6-month experimental limbs (2.4 vs. 4.7 Ncm/°, p < 0.0001). Our study suggests that the 6-month standard rabbit knee model of arthrofibrosis should continue to be used in the laboratory assessment of arthrofibrosis. However, the abbreviated model may be beneficial under selected experimental conditions.

AimsTo evaluate the role of autophagy in primary knee fibroblasts undergoing myofibroblast differentiation as an in vitro model of arthrofibrosis, a complication after total knee arthroplasty characterized by aberrant intra-articular scar tissue formation and limited range of motion.MethodsWe conducted a therapeutic screen of autophagic-modulating therapies in primary human knee fibroblasts undergoing transforming growth factor-beta 1 (TGF-β1)-mediated myofibroblast differentiation. Autophagy was induced pharmacologically with rapamycin or by amino acid deprivation. Picrosirius red staining was performed to quantify collagen deposition. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting were conducted to evaluate fibrotic gene expression levels.ResultsRapamycin, an mTOR complex 1 (mTORC1) inhibitor and autophagy inducer, reduced TGF-β1-mediated collagen deposition. Interestingly, we simultaneously report that myofibrogenic genes, including ACTA2, were highly upregulated following rapamycin-TGF-β1 treatment. When autophagy was induced through amino acid deprivation, we demonstrated suppressed extracellular matrix levels, fibrotic gene expression (e.g. ACTA2), and SMAD2 phosphorylation levels in TGF-β1-stimulated fibroblasts.ConclusionOur findings demonstrate that the induction of cellular autophagy suppresses TGF-β1-induced collagen deposition in primary human knee fibroblasts. Taken together, these data suggest that cellular autophagy may be prophylactic against the pathogenesis of arthrofibrosis.Cite this article: Bone Joint Res 2025;14(4):328–337.

AbstractAdipoRon is an adiponectin receptor 1, 2 (ADIPOR1 and ADIPOR2) agonist with potential antifibrotic effects. Whether AdipoRon can mitigate joint stiffness in a rabbit model of arthrofibrosis is unknown. We examined the efficacy of intravenous (IV) AdipoRon at mitigating contracture in a rabbit model of knee arthrofibrosis. Fifty‐six female New Zealand White rabbits were divided into three dosing groups: vehicle (dimethyl sulfoxide, DMSO), 2.5 mg/kg AdipoRon, and 5 mg/kg AdipoRon. AdipoRon, in DMSO, was administered IV preoperatively and for 5 days postoperatively (30 rabbits, Aim 1). AdipoRon was again dosed similarly after Kirschner wire (K‐wire) removal at 8 weeks (26 rabbits; Aim 2). The primary outcome of joint passive extension angle (PEA,°) was measured at 8, 10, 12, 16, and 24 weeks following index surgery. At 24 weeks, rabbits were euthanized and limbs were harvested to measure posterior capsular stiffness (N cm/°). In Aim 1, the 5 mg/kg treated rabbits had a significant increase in PEA when compared to controls at 16‐week (p < 0.05). In Aim 2, the 5 mg/kg treated rabbits had a significant increase in PEA when compared to controls at 10‐week (p < 0.05). In both aims, no significant differences were observed at later time points. Capsular stiffness was no different in any group. We are the first to report the efficacy of IV AdipoRon in a rabbit model of arthrofibrosis. We identified a significant dose‐dependent decrease in joint PEA at early time points; however, no differences were observed between groups at later time points. Clinical Significance: The present investigation provided the first assessment of AdipoRon's efficacy in mitigating knee stiffness in the current gold standard rabbit model of arthrofibrosis. Results of this investigation provided further evidence as to the potential role of AdipoRon as a preventative for arthrofibrosis in large mammals.

AbstractArthrofibrosis, which causes joint motion restrictions, is a common complication following total knee arthroplasty (TKA). Key features associated with arthrofibrosis include myofibroblast activation, knee stiffness, and excessive scar tissue formation. We previously demonstrated that adiponectin levels are suppressed within the knee tissues of patients affected by arthrofibrosis and showed that AdipoRon, an adiponectin receptor agonist, exhibited anti‐fibrotic properties in human mesenchymal stem cells. In this study, the therapeutic potential of AdipoRon was evaluated on TGFβ1‐mediated myofibroblast differentiation of primary human knee fibroblasts and in a mouse model of knee stiffness. Picrosirius red staining revealed that AdipoRon reduced TGFβ1‐induced collagen deposition in primary knee fibroblasts derived from patients undergoing primary TKA and revision TKA for arthrofibrosis. AdipoRon also reduced mRNA and protein levels of ACTA2, a key myofibroblast marker. RNA‐seq analysis corroborated the anti‐myofibrogenic effects of AdipoRon. In our knee stiffness mouse model, 6 weeks of knee immobilization, to induce a knee contracture, in conjunction with daily vehicle (DMSO) or AdipoRon (1, 5, and 25 mg/kg) via intraperitoneal injections were well tolerated based on animal behavior and weight measurements. Biomechanical testing demonstrated that passive extension angles (PEAs) of experimental knees were similar between vehicle and AdipoRon treatment groups in mice evaluated immediately following immobilization. Interestingly, relative to vehicle‐treated mice, 5 mg/kg AdipoRon therapy improved the PEA of the experimental knees in mice that underwent 4 weeks of knee remobilization following the immobilization and therapy. Together, these studies revealed that AdipoRon may be an effective therapeutic modality for arthrofibrosis.

The bone marrow microenvironment acts as a protective niche for malignant cells. Within the bone marrow microenvironment is the endosteal niche, which is the area where the ossified surface of the bone and marrow cavity interact. The endosteal niche provides the opportunity for hematological malignancies such as acute myeloid leukemia (AML) of the bone marrow to interact with osteoblasts from the bone. To study the interaction between AML cells and osteoblasts, our lab developed a co‐culture model of osteoblasts (MC3T3 osteoblast cell line) and AML cells (KG1a cell line or patient samples from bone marrow aspirates). AML cells expressing high levels of CXCR4 were cultured alone or with osteoblasts and then challenged with SDF‐1, the ligand to CXCR4 and a chemokine readily abundant in the bone marrow microenvironment that our lab has previously shown actually induces apoptosis in AML cells expressing high levels of CXCR4 and that osteoblasts are able to protect AML cells from SDF‐1 induced apoptosis. Our lab now shows that treating osteoblasts with cyclosporine (CSA) prevents their ability to effectively protect AML cells from SDF‐1 induced cell death as assayed by annexin‐V staining of AML cells by flow cytometry. CSA treatment of osteoblasts decreased protein levels of tissue non‐specific alkaline phosphatase (ALPL), a phosphatase necessary for osteoblast mineralization, compared to vehicle control. As such, osteoblasts were treated with control siRNA or ALPL siRNA. CXCR4 expressing AML cells were then co‐cultured with the transfected osteoblasts and challenged with SDF‐1. siRNA knockdown of ALPL in osteoblasts was sufficient to prevent protection of AML cells from the apoptotic stimuli. These studies indicate that osteoblast differentiation plays a role in mediating the protection of AML cells. Therefore, targeting specific differentiation factors in osteoblasts may help to make to bone marrow microenvironment a more hostile environment for AML cells and aid in better malignancy eradication. Support or Funding Information This study was supported by NIH R21 CA194217 (to K.E.H.). R.M.S. is supported by the Mayo Clinic Medical Scientist Training Program Robert L. Howell Physician‐Scientist Scholarship.

AbstractArthrofibrosis, which is characterized by excessive scar tissue and limited motion, can complicate the daily functioning of patients after total knee arthroplasty (TKA). Molecular hallmarks of arthrofibrosis include pathologic accumulation of myofibroblasts and disproportionate collagen deposition. Epigenetic mechanisms, including posttranslation modification of histones, control gene expression and may regulate fibrotic events. This study assessed the role of the bromodomain and extra‐terminal (BET) proteins on myofibroblast differentiation. This group of epigenetic regulators recognize acetylated lysines and are targeted by a class of drugs known as BET inhibitors. RNA‐seq analysis revealed robust mRNA expression of three BET members (BRD2, BRD3, and BRD4) while the fourth member (BRDT) is not expressed in primary TKA knee outgrowth fibroblasts. RT‐qPCR and western blot analyses revealed that BET inhibition with the small molecule JQ1 impairs TGFβ1‐induced expression of ACTA2, a key myofibroblast marker, in primary outgrowth knee fibroblasts. Similarly, JQ1 administration also reduced COL3A1 mRNA levels and collagen deposition as monitored by picrosirius red staining. Interestingly, the inhibitory effects of JQ1 on ACTA2 mRNA and protein expression, as well as COL3A1 expression and collagen deposition, were paralleled by siRNA‐mediated depletion of BRD4. Together, these data reveal that BRD4‐mediated epigenetic events support TGFβ1‐mediated myofibroblast differentiation and collagen deposition as seen in arthrofibrosis. To our knowledge, these are the first studies that assess epigenetic regulators and their downstream events in the context of arthrofibrosis. Future studies may reveal clinical utility for drugs that target epigenetic pathways, specifically BET proteins, in the prevention and treatment of arthrofibrosis.