Considering the Cellular Landscape in Marrow Stimulation Techniques for Cartilage Repair

Marrow stimulation is a common intervention used to treat cartilage defects in joints throughout the body and is characterized by the recruitment of bone marrow into the defects, theoretically initiating a regenerative process [1]. However, the repair tissue that results is often fibrocartilaginous in nature [2, 3], rather than hyaline cartilage, making it weaker and more susceptible to deterioration with time. This leads to poor repair tissue survivorship, leaving the entire joint at jeopardy of developing osteoarthritis (OA) [4]. Marrow stimulation has been employed in the repair of other musculoskeletal tissues, such as meniscus and rotator cuff [5, 6], yet it is still unclear why it does not consistently result in functional tissue restoration. The population of cells at the marrow stimulation site, and their contribution to inflammation, vascularity, and fibrosis, may play a role in repair tissue quality. Therefore, there is a need to understand the cellular landscape of bone marrow in the context of marrow stimulation techniques and to consider its potential role in cartilage repair.

Marrow stimulation, most commonly microfracture (MFx), involves perturbing the subchondral bone beneath the area of damaged cartilage, creating perforations 3–4 mm apart with an awl or a drill. When the subchondral plate is punctured, the underlying bone marrow and its cells are released into the cartilage defect, forming a “marrow clot” (Fig. 1a) [7]. The theory behind this approach is to recruit the pro-regenerative cells within the marrow, namely bone marrow mesenchymal stem cells (MSCs). MFx is primarily performed on patients below the age of 40, as cells tend to have diminished regenerative capacity past this age [1, 8]. Additionally, MFx is typically only recommended for patients with a defect smaller than 2 cm² [9]. Despite these limitations, an estimated 100,000 patients undergo MFx procedures per year, and improvements in current MFx techniques may even serve to expand this patient population [10]. The MFx approach has often disregarded the plethora of other cell types present in this repair environment, both those in the initial clot and those that migrate into the clot afterward (Fig. 1b). These cell types include many hematopoietic cells and immune cells, all of which may inform or inhibit new cartilage formation. The cell populations in bone marrow have been assessed via flow cytometry or RNA sequencing [11], mostly in regard to their contribution to marrow health and disease. However, the impact of these cell types, their ratios, and behaviors on cartilage repair is not known.

To improve the quality of repair following MFx, augmentation strategies with supplemental cells, biomaterials, and bioactive factors have been employed [12, 13]. Biomaterials have been found to play a significant role in MFx by enhancing the efficacy of repair, promoting tissue regeneration, and improving the long-term functional outcomes for individuals with cartilage defects [14‒16]. For example, known chondrogenic agents such as TGF-β3 and kartogenin (KGN) have improved both in vitro (in bone marrow MSCs) and in vivo (following MFx) cartilage tissue formation [17, 18], though recapitulation of native tissue properties remains elusive. Understanding how these augmentations impact the specific cell types that are recruited to the marrow stimulation site may be instrumental in guiding clinical progress. Thus, there is a need to investigate these augmentation modalities while considering the cellular landscape of the MFx environment.

In this review, we accomplish three goals: (1) to systematically review the clinical data on MFx augmentation with cells, materials, and bioactive factors to find assumptions on the biological elements recruited by MFx; (2) to review the cellular populations in bone marrow from single-cell RNA sequencing (scRNAseq) literature data and understand their impact on MFx healing; and (3) to highlight emerging technologies and approaches that could better guide these specific cell populations towards enhanced cartilage formation. We hope this review will guide future basic and clinical research to identify the right treatment approach to improve the efficacy of marrow stimulation to treat chondral, and other musculoskeletal, defects.

To better understand the cell types and elements assumed to be involved in MFx, we conducted a systematic review of clinical trials involving MFx on “clinicaltrials.gov” using the search terms “microfracture,” “marrow stimulation,” and “subchondral drilling” (search conducted July 2022). “Microfracture” and “subchondral drilling” were used in addition to “marrow stimulation” because these two terms are denoted as the main subtypes of marrow stimulation techniques. This search yielded 105 total trials (Fig. 2). Studies listed as suspended, terminated, or withdrawn, and studies of unknown status, were excluded from the search. Of these, studies that were not related to orthopedics were also excluded to focus on cartilage-related trials. This yielded 38 clinical trials that were used to extract data on assumed cell types and the different types of augmentations to standard MFx, including biomaterials and factors.

Half (19/38) of trials did not specify the type of cells or elements employed during MFx or marrow stimulation. The most common descriptions of cell/element types included “clots” (12 studies), “bone marrow” elements (n = 11), “blood” (n = 8), “stem cells” (n = 5), and “scar” (n = 3) (Fig. 3a, b). Of the described clots, clarifying adjectives included “marrow,” “fibrin,” “blood,” “well-anchored,” and “super.” “Bone marrow” elements included specific reference to mesenchymal stem or stromal cells only once and was mostly commonly utilized with “cells” alone. None of the trials discussed hematopoietic cells, macrophages, fibroblasts, or osteoblasts, though the description in many of the clinical trials was brief. Thus, the cellular makeup within bone marrow stimulation repair sites is not publicly reported in clinical trials, even though it likely represents an important player in MFx efficacy as a treatment for cartilage injuries, and in marrow stimulation as a treatment overall.

The most common approach in these clinical trials was to explore augmentations to MFx (21/38 = 55.3%), wherein standard MFx served as a control. An additional nine trials (23.7%) explored chondrocyte implantation techniques compared to MFx, and four (10.5%) investigated scaffold use without any form of marrow stimulation. Of the augmentation studies, thirteen explored biomaterials alone, two explored cells alone (including one synovial brushing for synovial cell recruitment), three explored biomaterials with cells, and two studies explored bone marrow aspirate concentrate, and one utilized a rehabilitation approach (bracing during recovery). The most common biomaterials included commercialized products (Table 1): BST-Cargel (chitosan bioscaffold implanted with autologous whole blood), Chondro-Gide (type I/III collagen membrane), and BioCartilage (micronized cartilage matrix). The supplementation of the MFx environment with additional cells included adipose-derived stem cells and umbilical cord blood-derived MSCs. Of note is that several of the clinical trials gave a generic purpose to their augmentation, for example, to “promote tissue regeneration,” “increase the amount and quality of quality cartilage repair,” provide “scaffolding for cellular growth,” and “encourage healing at the surgical site.” More specific modes of augmentation include “closest biomechanics and structure of normal cartilage,” “delivery of more specific progenitor cells,” and “stabilize the fragile blood clot that arises from microfracture.” These augmentations may have different effects on the various cell types recruited during marrow stimulation, complicating the clinical impacts of these of augmentations. A final consideration of these augmentations is the location; twenty-nine of the thirty-eight studies focused on the knee (including studies that specified femoral condyle, and femoral condyle-trochlea; Fig. 3c). Thus, prior clinical trials may have been restricted in their reports on the specific makeup of the cell types involved in MFx when developing new augmentation products, even though it may be influential in tissue formation and outcomes.

Bone marrow stimulation is often thought to rely on regenerative marrow elements or MSCs to heal cartilage defects. However, the MSC population makes up only a fraction of the bone marrow microenvironment [19, 20]. The formation of the MFx clot and subsequent recruitment of cells and regenerative factors involves a myriad of bone marrow resident cell types. The application of single-cell RNA sequencing (scRNAseq) has shed light on the bone marrow niche populations; however, providing a comprehensive list of all cell types in bone marrow, along with their estimated prevalence, is challenging due to variations in how each study tabulates this information [21‒26]. Understanding the role of these different cell populations and their contribution to vascularity, inflammation, fibrosis, and chondrogenesis may be vital to improving the outcomes of any cartilage repair intervention.

The scRNASeq data are quite broad and often prescreen cells with cluster of differentiation (CD) markers to isolate certain groups of cells. However, a few studies have been able to analyze the entire population (Fig. 4a). The predominant cell group is hematopoietic stem and progenitor cells (HSPCs; CD34+), containing a large portion of erythroblasts, and various progenitors for both immune and hematopoietic cells (e.g., neutrophils, monocytes, basophils, erythroblasts). A second population of cells are the differentiated immune cells, including neutrophils, lymphocytes (T cells, B cells, natural killer), and monocytes. Other cell types also include endothelial cells (arterial and sinusoidal) and Schwann cells (neuronal). The final group of cells is the mesenchymal cells, stromal cells with multiple lineages (Fig. 4b). This includes cells of adipogenic, osteogenic, and chondrogenic lineage, both progenitors and differentiated cells, as well as several types of fibroblasts (arteriolar, endosteal, stromal, myo-). Thus, the bone marrow microenvironment contains a broad range of cell types, and populations that change with age, disease, and co-morbidities [27, 28]. Below, we will further discuss these bone marrow resident populations and the mechanisms by which these cell types may modulate the outcome of marrow stimulation for cartilage repair.

HSPCs and immune cells have long been known to reside in the bone marrow [29, 30]. The HSPC population has a large population of erythroblasts, or red blood cell precursors, and megakaryocytes, or platelet producers. Platelets participate in repairing damage to blood vessels; they interact with coagulation factors and play a crucial role in hemostasis [31]. While cartilage is mostly avascular, MFx surgery causes ruptures in the vascularized subchondral bone, opening a path for platelet activation and infiltration. These platelets form a physical barrier in the form of a hemostatic plug that stabilizes the injury. The formation of a “marrow” clot provides a provisional fibrin-rich matrix for the recruited cells; additional MSCs and chondroprogenitor cells may migrate towards the fibrin mesh formed by platelet aggregation and participate in tissue repair [32]. The stability of the “marrow clot” derived from these platelets may be an important determinant of MFx outcomes. However, combined with the endothelial cells, there is a large influx of pro-angiogenic factors, including vascular endothelial growth factor (VEGF). This likely promotes the formation of new blood vessels, which can certainly aid in nutrient transport to the MFx site but may end up inhibiting chondrogenesis and functional cartilage repair.

The HSPC fraction also contains progenitor cell populations for neutrophils, granulocytes, and monocytes. Combined with the immune cell fraction, these cells typically coordinate the wound healing response. Neutrophils are among the first responders to injury [33, 34], and are likely recruited to the site of MFx, both from initial marrow flow into the defect and as a response to chemotactic signals produced by damaged cells. Neutrophils play a complex role in tissue repair and have been attributed to both positive and negative outcomes for cartilage repair. For example, studies have shown that applying biomaterials like deacetylated chitosan improves the chemotaxis of neutrophils to the MFx site and may promote tissue repair [35]. Neutrophils also release pro-inflammatory cytokines that lead to the recruitment of macrophages and natural killer (NK) cells [36]. These macrophages typically arise from monocytes and are classified into M1 and M2 subtypes [37]. M1 macrophages secrete pro-inflammatory cytokines such as IL-1β, TNF, and IL-6 [38] which inhibit MSC-mediated chondrogenic differentiation [36, 39]. Moreover, M1 macrophages may promote tissue fibrosis [40], which is a common MFx outcome, leading to inferior tissue quality. The M2 macrophage subtype, on the other hand, produces anti-inflammatory cytokines and pro-chondrogenic factors such as IL-10 and TGF-β, which promote the differentiation of MSCs into chondrocytes [36, 38]. Thus, the M1-M2 transition may be a key regulator towards improving repair.

The NK cells are another significant component of the body’s innate immune response that have been identified in the bone marrow [21, 23, 41] and may negatively influence cartilage repair by releasing interferon-γ (IFN-γ) [42], a cytokine that propels macrophages toward the M1 subtype [36, 43]. T and B lymphocytes make up the major immune cell fraction of the adaptive immune system and may play a crucial role in immunomodulation and cytokine production. For example, CD4 + T helper 1 (Th1) can produce IL-2 and IFN-γ which can activate macrophages, NK cells [44], and CD8 + cytotoxic T cells [45, 46], potentially inhibiting chondrogenesis. Thus, it may be necessary to consider these non-stromal cells and the likely impact they have on marrow-mediated cartilage repair.

The stromal population reported in the bone marrow by scRNAseq analysis includes osteoblasts, chondrocytes, MSCs, fibroblasts, and adipocytes [21, 22, 24‒26]. These cells may play important roles in wound healing by either directly participating in cartilage repair or by secreting growth factors, cytokines, and ECM components that promote tissue repair. Naturally, the chondrocytes and chondroprogenitors in bone marrow would be ideal cells to be recruited to the site, as they produce and maintain cartilage ECM [47] by depositing collagen and proteoglycans that are required to maintain the matrix [48, 49]. The stromal marrow is also rich in adipocytes and pre-adipocytes. In the past, adipocytes were mainly considered lipid storage depots but are now widely known for their endocrine functions. Adipocytes and pre-adipocytes can produce potent inflammatory mediators [50] that may negatively influence MSC-mediated cartilage repair. Conversely, adipose tissue is also a source of a subpopulation of MSCs known as adipose-derived mesenchymal stem cells (ADMSCs) that have demonstrated robust chondrogenic potential [51]. There are also several types of osteogenic cell types, potentially explaining pockets of calcified deposits in MFx defects, and thus their contribution to overall cartilage repair may be of importance [52]. Finally, fibroblasts are another significant population identified in the bone marrow that contributes to ECM synthesis. They are metabolically active cells that can release pro-inflammatory cytokines [53] that may inhibit chondrogenesis and promote a fibrotic phenotype [54]. Perhaps the most important types of marrow cells are the MSCs, which are recruited to the MFx clot, both from the initial marrow flow and in response to chemotactic signals. Here, they can differentiate into chondrocytes upon stimulation with chondrogenic factors like TGF-β [55]. Interestingly, MSCs can also push macrophages towards the anti-inflammatory M2 subtype [56], which could further enhance chondrogenic potential. While MSCs can certainly contribute to enhanced cartilage repair, they can also differentiate down other lineages (fibrotic, osteogenic, adipogenic) that may complicate chondrogenesis.

It is well known that the marrow cell environment changes significantly with age. Aged MSCs experience a decline in cell colony numbers, indicating a reduction in the overall amount of MSCs within the marrow. This decrease is accompanied by a notable attenuation of cell proliferation, which contributes to a diminished regenerative potential as the cells age [36, 57]. For example, the osteogenic abilities of aged MSCs are compromised, as evidenced through reduced alkaline phosphatase activity, diminished ECM mineralization, and an increased propensity for adipogenic differentiation [58]. The application of stem cell regenerative therapy has been found to be less effective in elderly individuals compared to their younger counterparts; one study by Gao et al. [59] demonstrated that bone regeneration was impaired in older mice after human muscle-derived stem cells were transplanted into the defect [57, 59]. Significantly, aging is known to induce cellular senescence, the cessation of cell division. Senescent MSCs have been found to be more sensitive to oxidative stress and DNA double strand breaks, as they exhibit reduced antioxidant capabilities and have been shown to downregulate the genes required for DNA damage repair [60]. Clinical application of MSCs is greatly affected by cell senescence, as MSCs with a high senescent population demonstrate reduced effectiveness in regenerative treatments upon injection [61]. These findings highlight the relationship between aging, cellular senescence, and the functional decline of MSCs in the marrow. Beyond the MSCs, the relative ratios and amounts of cells in the marrow change drastically with age, with adipose fractions approaching 70% by age 65 [62]. There are also decreases in B cell and T cell numbers, as well as metabolic dysfunction in HSPCs, with age [63, 64]. Beyond aging, co-morbidities also greatly impact the marrow cell composition. For example, diabetes can lead to reduced MSC numbers and accelerated adipogenesis [65]. Since many cartilage repair clinical trials are only performed on ideal patients (young, limited co-morbidities [13]), there is a need to consider the cellular landscape in aged/diseased marrow when translating MFx augmentations to these patients.

From our review of the marrow cell population, there appear to be several barriers to the formation of healthy cartilage repair tissue following MFx procedures. Insufficient populations of MSCs retained in the cartilage defect may hinder the regenerative capacity at the MFx site and can prevent regrowth of healthy hyaline tissue. For example, previous research has shown that about 7–10 MSCs per one million mononucleated cells can be isolated from bone marrow aspirate; therefore, a chondral defect undergoing MFx may contain fewer than 100 MSCs, whereas a comparable volume of cartilage would contain 10–20 million chondrocytes [66, 67]. Moreover, exposure to hematopoietic cells within the bone marrow may promote vascularity, which can be counterproductive considering cartilage’s inherently avascular nature. The vascularization of MFx defects is quite common and has been implicated in soft tissue fibrosis [68‒71]. Marrow stimulation also results in exposure to macrophages, which secrete pro-inflammatory cytokines and have been associated with the inhibition of chondrogenesis and the formation of fibrotic tissue [72, 73]. In light of these challenges, various emerging technologies are being explored to specifically address these obstacles and guide the process of cartilage regeneration; many of these advances are described below.

Though the exact amount is variable, there is generally a low content of MSCs in bone marrow. Without a sufficient number of MSCs in the marrow clot, the repair process is weakened, which may result in inferior tissue formation [74]. This may be mitigated by harvesting autologous MSCs to expand and deliver in augmentation to the MFx site [74, 75]. In older patients, stem cells are not only decreased in number, but they also become senescent. Antioxidants such as ascorbic acid, Cirsium setidens, and lactoferrin have been shown to alleviate cell senescence by inhibiting ROS production in MSCs [76]. Hypoxic culture has been found to inhibit MSC senescence, while simultaneously enhancing cell proliferation rate and differentiation ability as well as retaining stem cell properties [77]. Emerging research has also found that MSCs work well when supplemented with other cell types. When co-cultured with articular chondrocytes, MSCs release exosomes that prevent chondrocyte apoptosis and induce proliferation [78]. Clinical and animal studies have revealed that augmentation of bone marrow MSCs with other stem cells such as ADSCs and iPSCs leads to significantly improved outcomes post-MFx repair [79, 80]. Another promising avenue of research on MFx repair is the reprogramming of resident cells to be more cartilage-like, or to at least turn off their anti-chondrogenic phenotype. CRISPR/Cas9-mediated gene editing of human chondrocytes targeting MMP13 has recently been shown, in spheroid cultures, to reduce inflammation and enhance the expression of type II collagen, an important marker of healthy cartilage repair [81]. Further research shows that the use of CRISPR/Cas9 to ablate the genes encoding NGF, IL-1β and MMP13 in whole knee joints reduces joint pain and mitigates the progression of osteoarthritis in a mouse model [82]. Harnessing the regenerative potential of MSCs and the innovative applications of gene editing techniques, such as CRISPR/Cas9, offer new avenues for enhancing cartilage repair by reprogramming both exogenously added or endogenously recruited cells. These advancements may show promise for the development of more effective and minimally invasive treatments, improving outcomes for individuals suffering from cartilage degradation.

VEGF, a primary signaling driver of angiogenesis, is one of the most potent factors in endochondral bone formation and the pathology of joint diseases such as osteoarthritis (OA) [83]. VEGF regulates and drives blood vessel invasion from the metaphysis and existing endochondral bone into newly formed bone [84, 85]. Under normal conditions, articular cartilage does not express VEGF, but it is expressed in arthritic joints in both cartilage and synovium [86]. In related disease states such as rheumatoid arthritis, VEGF drives synovial angiogenesis, leading to inflammation and vascular infiltration of cartilage [87]. VEGF expression is increased by oxidative stress and hypoxia, mechanical stress, and inflammatory agents such as IL-1, TNF, IL-17 (Fig. 5a) [88].

Several studies have modulated VEGF signaling. For example, chondroitin sulfate (CS) reduces inflammation and cartilage catabolism [89], likely from its anti-angiogenic properties by counteracting IL-1β-dependent TSP-1 and VEGF downregulation at both protein and gene expression levels [90]. The inhibitor PPI 2458 also reduces synovial and osteochondral angiogenesis and subsequent inflammation, osteophyte formation, and joint pain in rodent models via VEGF arrest [91]. Additionally, the humanized anti-VEGF monoclonal antibody bevacizumab has demonstrated preclinical success in the mitigation of OA [92], and specifically in cartilage repair, has enhanced chondrogenesis and reduced angiogenesis when embedded in a fibrin/hyaluronan scaffold for controlled temporal release [93]. Similar scaffold therapies with conjugated anti-angiogenic drugs have gained traction in recent years. SFtt-1 (Soluble Fms-like tyrosine kinase-1, a VEGF antagonist) has been used in stem cell therapy studies, paired with muscle-derived stem cells (MDSCs) to obtain a positive impact on chondrogenic differentiation [94]. The use of plasmid vectors encoding endogenous angiogenesis inhibitors in cell populations seeded within implanted scaffolds may be a strategy to maintain engineered cartilage phenotypes in vivo [95]. Finally, one interesting approach in the cartilage repair space is to apply platelet-rich plasma (PRP) to the cartilage repair site, introducing a plethora of pro-healing growth factors. However, since PRP also contains VEGF, a recent study utilized a VEGF-binding system to attenuate the concentration of VEGF in PRP, and even showed that this enhanced cartilage repair in rodents (Fig. 5b) [96]. While it seems disrupting VEGF has promising results, such strategies must be further studied to understand and balance their long-term impact on tissue repair of cartilage and overall joint homeostasis. The ideal administration of these therapies points towards their local application to the site of MFx to establish an avascular environment via the inhibition of VEGF signaling.

Macrophages are a critical component of the immune system that are found both in marrow and in the synovial lining, and they perform distinct functions based on their M1 versus M2 polarity (Fig. 6). In general, M1 macrophages are associated with impeding the proliferation and immunosuppressive capabilities of MSCs, as well as their chondrogenic potential. M2 macrophages, however, are linked to the promotion of wound healing via pro-chondrogenic cytokines like IL-10, IL-1RA, and TGF-β. One method to guide this is physical exercise, which can promote the polarization of macrophages towards the M2 phenotype by stimulating a type 2 immune response and altering the synovial microenvironment. Type II collagen can increase the expression of genes linked to the M2 phenotype, causing an increase in TGF-β production [72]. Another route involves M2 macrophage-derived exosomes to enhance the repair of acellular cartilage extracellular matrix (ACECM) scaffolds [97]. In vitro, these scaffolds show enhanced proliferation, migration, and chondrogenic differentiation of BMSCs while also promoting M2 polarization. In vivo, the ACECM scaffold enhanced osteochondral regeneration and helped regulate the inflammatory environment of the joint. Interestingly, a recent study employed M1, M1-like, M2, and M2-like macrophages for the cultivation of cartilage pellets, showing the M1-like macrophages provided the best chondrogenesis [98]. However, it was also noted that the M1-like macrophages had converted to a more M2-like phenotype, indicating that guiding this polarity has large impacts on chondrogenesis. Studies have shown that MSCs may inhibit M1 macrophages and promote M2 macrophages in vitro, demonstrating the potential of exogenous MSC application to MFx sites in improving cartilage repair from an immunomodulatory standpoint. MSCs secrete many anti-inflammatory molecules, such as IL-10, TGF-β, PGE2, IDO, NO, and FAS/FASL, that diminish pro-inflammatory cytokine release and inhibit the function of immune cells, suppressing the body’s response to local inflammation [99, 100]. MSCs also exhibit anti-apoptotic activity via paracrine signaling, which protects damaged cells and creates a regeneration-privileged environment.

Marrow stimulation has gained significant traction as a repair procedure and has thus been expanded to tissues beyond cartilage. For example, ankle fusion is one of the most common procedures for treating end-stage osteoarthritis of the foot and ankle and involves joint debridement, marrow drilling, and screw and/or plate fixation [101]. Since the ankle environment is notoriously difficult for healing and lacks sufficient vascularity compared to other fusion sites [102], marrow stimulation is thought to be required to achieve successful foot and ankle fusion. Saad et al. [103] reported that the concentration of MSCs within the bone marrow can greatly affect bony union, further highlighting that the low content of MSC’s in marrow may need to be supplemented to enhance healing. Unlike the cartilage repair needs described previously, the introduction of vascularity into fusion environments is typically necessary. Thus, enhancing VEGF-mediated angiogenesis may be seen as a positive, as would the osteoblasts and osteoprogenitors and their contribution to healing. Another trending application of marrow stimulation is for augmented rotator cuff repair. Rotator cuff injuries are difficult to heal due to the injury site and tendon size, and the fibrous scar tissue that forms after repair is disorganized. Furthermore, repair fails to recapitulate the enthesis or the fibrocartilaginous transition zone between bone and tendon. This leads to increased biomechanical instability and a decreased loading potential [104]. A systematic review published in 2023 by Zhang et al. found that marrow stimulation during arthroscopic repair of the rotator cuff significantly reduced retear rates after 2 years [105]. However, ROM and other orthopedic evaluation scores (VAS, UCLA, ASES, DASH) did not yield significant differences. These results are verified and validated by studies done by Ajrawat and Li [106, 107]. From these papers, it is clear that marrow stimulation can assist clinical healing, though understanding and guiding the heterogenous marrow cell population for the specific intended application may be paramount.

This review provides an analysis and interpretation of the cellular landscape of marrow stimulation for cartilage repair, emphasizing the need for a more nuanced understanding of cellular heterogeneity. The systematic review of clinical trials revealed oversights in specifying the types of cells or elements employed during MFx, with only half of the trials providing such information. The review of bone marrow cell populations demonstrated a highly heterogeneous mix, including hematopoietic cells, osteoblasts, fibroblasts, macrophages, and only a small population of MSCs. Understanding the roles of these cell populations in vascularity, inflammation, fibrosis, and chondrogenesis is crucial for improving the outcomes of cartilage repair interventions. In light of the challenges identified in current MFx techniques, emerging technologies were discussed to address these obstacles. Strategies to account for low stem cell numbers, modulate VEGF-mediated vascularization, and guide macrophage polarity for improved cartilage formation were explored. These advancements, including the use of gene editing techniques like CRISPR/Cas9, anti-angiogenic therapies, and immunomodulatory approaches, offer promising avenues to enhance cartilage repair. By addressing the heterogeneity of cell populations and implementing innovative approaches, future research and clinical practice can strive towards more successful and durable MFx outcomes.

The authors thank the Department of Orthopaedics at Emory University for their support.

M.H., L.M.F., and J.M.P. are co-inventors on a PCT patent application. J.M.P. is a consultant for NovoPedics Inc. and a co-founder of Forsagen LLC. These potential conflicts did not influence the writing of this manuscript.

This review was supported by a literature grant (21–273) from the ON Foundation, Switzerland. The funder had no role in the design, data collect, data analysis, and reporting of this study.

M.H. and J.M.P. performed and interpreted a systematic review of clinical trials. M.H., L.M.F., H.S., T.P., N.L.H., S.A.P., and J.M.P. performed literature review, and formulation and drafting of the manuscript sections. M.H., L.M.F., H.S., T.P., N.L.H., S.A.P., J.T.B., J.M.K., and J.M.P. contributed to writing and editing the manuscript.