UNASSIGNED: Several tissues contribute to the onset and advancement of knee osteoarthritis (OA). One tissue type that is worthy of closer evaluation, particularly in the context of sex, is the infrapatellar fat pad (IFP). We previously demonstrated that removal of the IFP had short-term beneficial effects for a cohort of male Dunkin-Hartley guinea pigs. The present project was designed to elucidate the influence of IFP removal in females of this OA-prone strain. It was hypothesized that resection of the IFP would reduce the development of OA in knees of a rodent model predisposed to the disease.
UNASSIGNED: Female guinea pigs (n=16) were acquired at an age of 2.5 months. Surgical removal of the IFP and associated synovium complex (IFP/SC) was executed at 3 months of age. One knee had the IFP/SC resected; a comparable sham surgery was performed on the contralateral knee. All animals were subjected to voluntary enclosure monitoring and dynamic weight-bearing, as well as compulsory treadmill-based gait analysis monthly; baseline data was collected prior to surgery. Guinea pigs were euthanized at 7 months. Knees from eight animals were evaluated via histology, mRNA expression, and immunohistochemistry (IHC); knees from the remaining eight animals were allocated to microcomputed tomography (microCT), biomechanical analyses (whole joint testing and indentation relaxation testing), and atomic absorption spectroscopy (AAS).
UNASSIGNED: Fibrous connective tissue (FCT) replaced the IFP/SC. Mobility/gait data indicated that unilateral IFP/SC removal did not affect bilateral hindlimb movement. MicroCT demonstrated that osteophytes were not a significant feature of OA in this sex; however, trabecular thickness (TbTh) in medial femorae decreased in knees containing the FCT. Histopathology scores were predominantly influenced by changes in the lateral tibia, which demonstrated that histologic signs of OA were increased in knees containing the native IFP/SC versus those with the FCT. Similarly, indentation testing demonstrated higher instantaneous and equilibrium moduli in the lateral tibial articular cartilage of control knees with native IFPs. AAS of multiple tissue types associated with the knee revealed that zinc was the major trace element influenced by removal of the IFP/SC.
UNASSIGNED: Our data suggest that the IFP/SC is a significant component driving knee OA in female guinea pigs and that resection of this tissue prior to disease has short-term benefits. Specifically, the formation of the FCT in place of the native tissue resulted in decreased cartilage-related OA changes, as demonstrated by reduced Osteoarthritis Research Society International (OARSI) histology scores, as well as changes in transcript, protein, and cartilage indentation analyses. Importantly, this model provides evidence that sex needs to be considered when investigating responses and associated mechanisms seen with this intervention.

During the progression of knee osteoarthritis (OA), the synovium and infrapatellar fat pad (IFP) can serve as source for Substance P (SP) and calcitonin gene-related peptide (CGRP), two important pain-transmitting, immune, and inflammation modulating neuropeptides. Our previous studies showed that infrapatellar fat pad-derived mesenchymal stem/stromal cells (MSC) acquire a potent immunomodulatory phenotype and actively degrade Substance P via CD10 both in vitro and in vivo. On this basis, our hypothesis is that CD10-bound IFP-MSC sEVs can be engineered to target CGRP while retaining their anti-inflammatory phenotype. Herein, human IFP-MSC cultures were transduced with an adeno-associated virus (AAV) vector carrying a GFP-labelled gene for a CGRP antagonist peptide (aCGRP). The GFP positive aCGRP IFP-MSC were isolated and their sEVs\' miRNA and protein cargos were assessed using multiplex methods. Our results showed that purified aCGRP IFP-MSC cultures yielded sEVs with cargo of 147 distinct MSC-related miRNAs. Reactome analysis of miRNAs detected in these sEVs revealed strong involvement in the regulation of target genes involved in pathways that control pain, inflammation and cartilage homeostasis. Protein array of the sEVs cargo demonstrated high presence of key immunomodulatory and reparative proteins. Stimulated macrophages exposed to aCGRP IFP-MSC sEVs demonstrated a switch towards an alternate M2 status. Also, stimulated cortical neurons exposed to aCGRP IFP-MSC sEVs modulate their molecular pain signaling profile. Collectively, our data suggest that yielded sEVs can putatively target CGRP in vivo, while containing potent anti-inflammatory and analgesic cargo, suggesting the promise for novel sEVs-based therapeutic approaches to diseases such as OA.

OBJECTIVE: Osteoarthritis (OA) is a whole-joint disease in which the role of the infrapatellar fat pad (IFP) in its pathogenesis is unclear. Our study explored the cellular heterogeneity of IFP to understand OA and identify therapeutic targets.
METHODS: Single-cell and single-nuclei RNA sequencing were used to analyze 10 IFP samples, comprising 5 from OA patients and 5 from healthy controls. Analyses included differential gene expression, enrichment, pseudotime trajectory, and cellular communication, along with comparative studies with visceral and subcutaneous fats. Key subcluster and pathways were validated using multiplex immunohistochemistry.
RESULTS: The scRNA-seq performed on the IFPs of the OA and control group profiled the gene expressions of over 49,674 cells belonging to 11 major cell types. We discovered that adipose stem and progenitor cells (ASPCs), contributing to the formation of both adipocytes and synovial-lining fibroblasts (SLF). Interstitial inflammatory fibroblasts (iiFBs) were a subcluster of ASPCs that exhibit notable pro-inflammatory and proliferative characteristics. We identified four adipocyte subtypes, with one subtype showing a reduced lipid synthesis ability. Furthermore, iiFBs modulated the activities of macrophages and T cells in the IFP. Compared to subcutaneous and visceral adipose tissues, iiFBs represented a distinctive subpopulation of ASPCs in IFP that regulated cartilage proliferation through the MK pathway.
CONCLUSIONS: This study presents a comprehensive single-cell transcriptomic atlas of IFP, uncovering its complex cellular landscape and potential impact on OA progression. Our findings highlight the role of iiFBs in OA, especially through MK pathway, opening new avenues for understanding OA pathogenesis and developing novel targeted therapies.

The onset and progression of human inflammatory joint diseases are strongly associated with the activation of resident synovium/infrapatellar fat pad (IFP) pro-inflammatory and pain-transmitting signaling. We recently reported that intra-articularly injected IFP-derived mesenchymal stem/stromal cells (IFP-MSC) acquire a potent immunomodulatory phenotype and actively degrade substance P (SP) via neutral endopeptidase CD10 (neprilysin). Our hypothesis is that IFP-MSC robust immunomodulatory therapeutic effects are largely exerted via their CD10-bound small extracellular vesicles (IFP-MSC sEVs) by attenuating synoviocyte pro-inflammatory activation and articular cartilage degradation. Herein, IFP-MSC sEVs were isolated from CD10High- and CD10Low-expressing IFP-MSC cultures and their sEV miRNA cargo was assessed using multiplex methods. Functionally, we interrogated the effect of CD10High and CD10Low sEVs on stimulated by inflammatory/fibrotic cues synoviocyte monocultures and cocultures with IFP-MSC-derived chondropellets. Finally, CD10High sEVs were tested in vivo for their therapeutic capacity in an animal model of acute synovitis/fat pad fibrosis. Our results showed that CD10High and CD10Low sEVs possess distinct miRNA profiles. Reactome analysis of miRNAs highly present in sEVs showed their involvement in the regulation of six gene groups, particularly those involving the immune system. Stimulated synoviocytes exposed to IFP-MSC sEVs demonstrated significantly reduced proliferation and altered inflammation-related molecular profiles compared to control stimulated synoviocytes. Importantly, CD10High sEV treatment of stimulated chondropellets/synoviocyte cocultures indicated significant chondroprotective effects. Therapeutically, CD10High sEV treatment resulted in robust chondroprotective effects by retaining articular cartilage structure/composition and PRG4 (lubricin)-expressing cartilage cells in the animal model of acute synovitis/IFP fibrosis. Our study suggests that CD10High sEVs possess immunomodulatory miRNA attributes with strong chondroprotective/anabolic effects for articular cartilage in vivo. The results could serve as a foundation for sEV-based therapeutics for the resolution of detrimental aspects of immune-mediated inflammatory joint changes associated with conditions such as osteoarthritis (OA).

To investigate the in vitro and in vivo anti-inflammatory/anti-fibrotic capacity of IFP-MSC manufactured as 3D spheroids. Our hypothesis is that IFP-MSC do not require prior cell priming to acquire a robust immunomodulatory phenotype in vitro in order to efficiently reverse synovitis and IFP fibrosis, and secondarily delay articular cartilage damage in vivo.
Human IFP-MSC immunophenotype, tripotentiality, and transcriptional profiles were assessed in 3D settings. Multiplex secretomes were assessed in IFP-MSC spheroids [Crude (non-immunoselected), CD146+ or CD146- immunoselected cells] and compared with 2D cultures with and without prior inflammatory/fibrotic cell priming. Functionally, IFP-MSC spheroids were assessed for their immunopotency on human PBMC proliferation and their effect on stimulated synoviocytes with inflammation and fibrotic cues. The anti-inflammatory and anti-fibrotic spheroid properties were further evaluated in vivo in a rat model of acute synovitis/fat pad fibrosis.
Spheroids enhanced IFP-MSC phenotypic, transcriptional, and secretory immunomodulatory profiles compared to 2D cultures. Further, CD146+ IFP-MSC spheroids showed enhanced secretory and transcriptional profiles; however, these attributes were not reflected in a superior capacity to suppress activated PBMC. This suggests that 3D culturing settings are sufficient to induce an enhanced immunomodulatory phenotype in both Crude and CD146-immunoselected IFP-MSC. Crude IFP-MSC spheroids modulated the molecular response of synoviocytes previously exposed to inflammatory cues. Therapeutically, IFP-MSC spheroids retained substance P degradation potential in vivo, while effectively inducing resolution of inflammation/fibrosis of the synovium and fat pad. Furthermore, their presence resulted in arrest of articular cartilage degradation in a rat model of progressive synovitis and fat pad fibrosis.
3D spheroids confer IFP-MSC a reproducible and enhanced immunomodulatory effect in vitro and in vivo, circumventing the requirement of non-compliant cell priming or selection before administration and thereby streamlining cell products manufacturing protocols.

Synovitis and infrapatellar fat pad (IFP) fibrosis participate in various conditions of the knee. Substance P (SP), a neurotransmitter secreted within those structures and historically associated with nociception, also modulates local neurogenic inflammatory and fibrotic responses. Exposure of IFP mesenchymal stem cells (IFP-MSCs) to a proinflammatory/profibrotic environment (ex vivo priming with TNFα, IFNγ, and CTGF) induces their expression of CD10/neprilysin, effectively degrading SP in vitro and in vivo.
The purpose was to test the therapeutic effects of IFP-MSCs processed under regulatory-compliant protocols, comparing them side-by-side with standard fetal bovine serum (FBS)-grown cells. The hypothesis was that when processed under such protocols, IFP-MSCs do not require ex vivo priming to acquire a CD10-rich phenotype efficiently degrading SP and reversing synovitis and IFP fibrosis.
Controlled laboratory study.
Human IFP-MSCs were processed in FBS or either of 2 alternative conditions-regulatory-compliant pooled human platelet lysate (hPL) and chemically reinforced medium (Ch-R)-and then subjected to proinflammatory/profibrotic priming with TNFα, IFNγ, and CTGF. Cells were assessed for in vitro proliferation, stemness, immunophenotype, differentiation potential, transcriptional and secretory profiles, and SP degradation. Based on a rat model of acute synovitis and IFP fibrosis, the in vivo efficacy of cells degrading SP plus reversing structural signs of inflammation and fibrosis was assessed.
When compared with FBS, IFP-MSCs processed with either hPL or Ch-R exhibited a CD10High phenotype and showed enhanced proliferation, differentiation, and immunomodulatory transcriptional and secretory profiles (amplified by priming). Both methods recapitulated and augmented the secretion of growth factors seen with FBS plus priming, with some differences between them. Functionally, in vitro SP degradation was more efficient in hPL and Ch-R, confirmed upon intra-articular injection in vivo where CD10-rich IFP-MSCs also dramatically reversed signs of synovitis and IFP fibrosis even without priming or at significantly lower cell doses.
hPL and Ch-R formulations can effectively replace FBS plus priming to induce specific therapeutic attributes in IFP-MSCs. The resulting fine-tuned, regulatory-compliant, cell-based product has potential future utilization as a novel minimally invasive cell therapy for the treatment of synovitis and IFP fibrosis.
The therapeutic enhancement of IFP-MSCs manufactured under regulatory-compliant conditions suggests that such a strategy could accelerate the time from preclinical to clinical phases. The therapeutic efficacy obtained at lower MSC numbers than currently needed and the avoidance of cell priming for efficient results could have a significant effect on the design of clinical protocols to potentially treat conditions involving synovitis and IFP fibrosis.

Recent studies have shown evidence that higher adiposity in the infrapatellar fat pad (IFP) induces inflammatory phenotypes in the knee joint and thereby contributes to the development and progression of osteoarthritis (OA). In particular, IFP adipocyte-derived inflammatory cytokines participate in pathological events. Our previous research has already addressed the therapeutic efficacy of hyaluronic acid and platelet-rich plasma (HA+PRP), including the promotion of cartilage regeneration and the inhibition of inflammation. The current study aimed to explore the remedial action of coadministered HA+PRP in OA recovery via IFP adipocyte inhibition.
HA+PRP repairs OA articular cartilage by inhibiting the release of adipokines from IFP adipocytes.
Controlled laboratory study.
IFP adipocytes and articular chondrocytes were obtained from 10 patients with OA, and the effects of releasates containing cytokines and adipokines in IFP adipocyte-derived conditioned medium (IACM) on articular chondrocytes and IFP adipocytes themselves were evaluated. The therapeutic efficacy of exogenous HA+PRP was determined through its administration to cocultured IFP adipocytes and articular chondrocytes and further demonstrated in a 3-dimensional (3D) arthritic neocartilage model.
The IACM and IFP adipocyte-induced microenvironment could induce dedifferentiated and inflammatory phenotypes in articular chondrocytes. HA+PRP decreased the inflammatory potential of IFP adipocytes through the profound inhibition of cytokines and adipokines. The IACM-mediated and -reduced cartilaginous extracellular matrix could also be recovered through HA+PRP in the 3D arthritic neocartilage model.
IFP adipocyte-derived releasates mediated inflammatory response dedifferentiation in chondrocytes, which was recovered through HA+PRP administration.
Our findings demonstrated that HA+PRP effectively diminished IFP adipocyte-promoted inflammation in articular chondrocytes, indicating that the IFP could be a potential therapeutic target for OA therapy.