2. The Bone Marrow Niche

The bone marrow (BM) is a semisolid tissue located in the central cavities of axial and long bones. This organ contains numerous cellular and molecular components. Typical examples of cell populations residing in the bone marrow can be divided into nonhematopoietic (e.g., pericytes, endothelial cells, osteoblasts, adipocytes and Schwann cells) and hematopoietic (e.g., neutrophils, lymphocytes, megakaryocytes, monocytes and osteoclasts) ^[7]. All of these cells and associated molecular profiles play important roles in the regulation of the bone marrow niche ^[8]. In addition to these cell types, there is also the presence of hematopoietic stem cells (HSC) and mesenchymal stem cells (MSC), the two major adult stem cell types in the bone marrow. These adult stem cells are highly praised for their ability to perform self-renewal and differentiation into specific mature cell lineages ^[9].

Hematopoietic stem cells are comprised of adult progenitor stem cells which are responsible for performing hematopoiesis. In other words, HSCs give rise to all of the blood cells. These cells are present in very small concentrations within the BM; the proportion is approximately 1:10,000 (one HSC for every ten thousand BM mononuclear cells) ^[10]. The mesenchymal stem cell is the other major adult stem cell type. It is characterized by a set of specific cell surface differentiation markers (CDs). MSCs are known to express a range of cell-lineage specific antigens which differ depending on culture preparation, duration, or plating density ^[11][12]. Previously, the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy (ISCT) proposed minimal characterization criteria for MSCs, which encompass the following attributes: the cells must be plastic-adherent while in culture; must be able to undergo differentiation in vitro into adipocyte, chondroblast, and osteoblast lineages; must express CD105, CD73 and CD90; must lack expression of CD45, CD11b, CD34 or CD14, CD79α or CD19 and HLA-DR surface markers ^[13].

MSCs are able to differentiate into mesodermal lineage cells such as cartilage, bone, fat, muscle, meniscus and tendon—a fundamental attribute in regenerative medicine ^[14]. Additionally, these cells are known for their paracrine effects, being able to manipulate their local microenvironment ^[15]. These cells are highly advantageous since they do not appear to trigger aggressive immunogenic episodes and can be easily isolated, enabling allogenic transplantation. In such circumstances, these cells should be considered immune-evasive. However, the effects of MSCs in cellular-based therapies tend to be more inclined toward their homing and engraftment abilities into target tissues ^[16]. It has been hypothesized that MSCs have a rather short life span and are phagocytized by monocytes, subsequently stimulating the production of T-regulatory cells. This sequence of events may very well contribute to overall clinical improvement ^[17].

Whenever tissue injury occurs, involved cells secrete chemical signals (chemokines) responsible for MSC recruitment. Once recruited into the target tissue, MSCs respond by modulating the wound-healing cascade, reducing apoptosis and fibrosis, attenuating the inflammatory process and stimulating cell proliferation and differentiation via paracrine and autocrine pathways ^[18]. These effects are due to the ability of MSCs to release key agents, including vasculoendothelial growth factor (VEGF), transforming growth factor beta (TGF-β), stromal-derived factor 1 (SDF-1), and stem cell factor (SCF), among others. Moreover, they can downregulate synthesis of interleukin (IL)-1, IL-6, interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), which are major pro-inflammatory cytokines ^[19][20][21]. MSCs also display immunomodulatory properties; they are able to inhibit the activation of type 1 macrophages, natural killer (NK) cells, and both B and T lymphocytes ^[22].

Researchers have also zoomed in on interleukin-1 receptor antagonist (IL-1RA), an important cytokine found in bone marrow-derived products. This protein is a competitive antagonist that binds to IL-1B and IL-1a isoforms of cell surface receptors and inhibits IL-1-induced catabolic reactions and inflammatory effects ^[23]. IL-1Ra has the potential to reduce matrix degradation, as IL-1B reportedly induces MMP-3 and TNF-a gene expression, prostaglandin E2 secretion, chondrocyte apoptosis, and inhibition of collagen deposition ^[24].

3. Bone Marrow Aspirate–Technique and Content

Aspiration of the bone marrow provides a rich source of several cellular and molecular components that elicit numerous effects on tissue regeneration, all of which are particularly beneficial for cartilage, bone and soft tissue injury ^[25]. This orthobiologic product is usually recommended for the treatment of specific musculoskeletal disorders, e.g., joint arthritis, bone defects, ligament tears, osteonecrosis of the femoral head, nonunion ^[26]. Bone marrow (BM), and consequently BMA, contains mesenchymal stem cells (MSCs), hematopoietic stem cells (HSCs), endothelial progenitor cells, and other progenitor cells—together with growth factors, including bone morphogenetic proteins (BMP), platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), and IL-1 receptor antagonist ^[27].

BMA can typically be harvested from different sites such as the iliac crest, proximal and distal tibia, or calcaneus. However, there is a certain preference to aspirate the bone marrow from the posterior iliac crest, because it has been found that this anatomical region offers a considerable amount of biological material with greater number of osteoblastic progenitors, compared to the tibia and calcaneus ^[28]. Additionally, different harvesting techniques have been experimented with, generating heterogeneity among reported results in regards to the number of progenitor cells ^[29]. The aspiration of small volumes of product in different subcortical areas appears to be more efficient, as it yields greater numbers of stem cell progenitors ^[30]. Medical practitioners may consider the utilization of fluoroscope or ultrasound to guide the aspiration procedure. The use of anatomic landmarks for the average patient and ultrasound guidance for larger individuals may also be recommended. For the average patient, an ideal volume between 90 and 120 milliliters should be drawn, whereas in a large individual, volumes greater than 120–150 mL should be avoided ^[31][32].

It is important to note that the majority of MSCs lie within subcortical areas, and pericytes tend to be located around the blood vessels (as their name indicates). Therefore, perforating the bone marrow cavity too deeply may not be a wise strategy. It will not yield the greatest amounts of target cells ^[31]. Figure 1 further illustrates BMA preparation techniques.

Figure 1. Bone marrow aspirate (BMA) Matrix. The combination of BMA with hyaluronic acid (HA) enables the formation of the BMA matrix, proposing a novel strategy towards the enhancement of regenerative mechanisms and possible synergistic effects. The crosslinked fibrin fibers derived from the BMA clot form a natural scaffold, supporting cellular activity and reconstruction of damaged tissue.

In regards to contraindications, patients with significant anemia, systemic infection, active hematologic neoplasm, or individuals who are unable to be anatomically positioned for the procedure should consider other alternatives. If the patient is making use of certain medications, these should be disclosed before the procedure. Examples include: prednisone, since it is known to elicit antianabolic effects; statins, which negatively impact cell proliferation; and nonsteroidal anti-inflammatory drugs, due to their interference in platelet aggregation and clot function ^[33].

4. Coagulated BMA–Potential Applications

The aspirated bone marrow tends to suffer rapid coagulation due to the presence of megakaryocytes, platelets and coagulation factors ^[34]. Surgeons may circumvent this inevitable event by coating the inside of the syringes with anticoagulant agents, usually acid citrate dextrose solution (ACD) or heparin solutions ^[35]. Although clot formation may be frowned upon and seen as a technical complication (with good reason), some researchers argue in favor of this outcome. From a biological standpoint, the clot may uncover positive attributes related to the therapeutic value of BMA itself and perhaps further enhance regenerative mechanisms ^[6]. Thinking critically, adding too many chemicals or exogenous products to the BMA product might interfere in its natural biological potential. Additionally, in order to cause coagulation, platelet degranulation is a required step; this step delivers numerous osteotropic cytokines and growth factors into the site of injury ^[32]. Another fundamental effect that is often overlooked is the fibrinolytic activity. The fibrinolytic cascade ultimately culminates in fibrin scissions, generating additional factors that are highly beneficial towards cell engraftment ^[36]. Della Bella et al. ^[37] illustrated these concepts by attempting to culture BMA in its coagulated and noncoagulated states. Fifteen days post-culture, the researchers observed that the BMA clot culture displayed higher growth kinetics of MSCs in comparison to the samples treated with anticoagulants. Yao and colleagues demonstrated the potential advantages of using bone marrow clots associated with three-dimensional biodegradable scaffolds in promoting extracellular matrix (ECM) scaffold chondrogenic regeneration ^[38]. It is important to note that due to the abundant presence of fibrin and erythrocytes in clots, there is a certain risk for an initial obstruction of nutrient exchange, which may cause some decrease in the ratio of cell viability. However, it was reported that the same scaffolds led to expressive improvements in cell adhesion, proliferation, and chondrogenic differentiation with porous recanalization in later cultures in comparison to other groups. Overall, there were superior results—in terms of DNA content, expression of Sox9 and RunX2 genes (both highly expressed during chondrocyte and osteoblast proliferation and differentiation), cartilage lacuna-like cells, and ECM accumulation—as well as greater scaffold size and mechanic stability in bone marrow clot cultures. In similar fashion, Pascher et al. conducted an appreciable in vitro study confirming that coagulates formed from BMA may be a useful gene delivery tool for cartilage and possibly other musculoskeletal structures—because it is possible to modify cells within the fluid with an adenoviral vector. Additionally, the clot-derived matrix is completely natural, native to the host, and is the fundamental platform for the healing and repair of mesenchymal tissues ^[39].

Perhaps one of the most interesting (and sometimes overlooked) features of coagulates is the presence and biological value of fibrin, which is the main component in these hematological manifestations—be it from peripheral blood or bone marrow. The fibrin clot that is formed after an injury can act as a shield to preserve tissues against blood loss and invasion of pathogens and may very well provide a temporary matrix (Figure 1) through which cells can migrate during the repair process ^[40][41]. Fibrin clots are composed of an agglomeration of platelets embedded in a network of crosslinked fibrin fibers as a result of the cleavage of fibrinogen by serine proteases. This promotes the polymerization of fibrin monomers—a primary event in blood coagulation ^[42]. This biological structure may also serve as a reservoir of cytokines and growth factors, which are released once activated platelets undergo degranulation ^[42]. An in vitro study led by Colley et al. ^[43] concluded that MSCs cultured on fibrin gels display enhanced proliferation potential and are capable of maintaining their osteogenic lineage differentiation potential when compared to MSCs cultured on plastic plates, suggesting that the fibrin matrix could help in maintaining stemness. In further advances, a clinical study ^[44] revealed optimistic results with the implantation of autologous MSC/fibrin clot constructs in patients suffering from upper limb nonunions. It was argued that the malleable fibrin gel texture in the clot facilitated diffusion of nutrients and differentiation of MSCs toward osteogenic lineage, encouraging tissue integration and healing. This strategy also showed significant safety and effectiveness in the long-term follow up of patients, since there were no occurrences of ectopic neoformation, neoplastic transformation, infection, overgrowth, or refracture. Although complex, researchers believe that the regenerative effects mediated by the clot tend to closely mimic the natural processes that take place in fracture hematomas.

Elaborating further, a BMA clot has superior advantages over other biological products; for instance, the bone marrow fibrin clot appears to offer a significantly higher concentration of VEGF, bFGF, IGF-1, SDF-1, and HGF (which are vital for cell survival, migration, differentiation, and regeneration) in exposed cell cultures than in cells treated with peripheral blood fibrin clot ^[45]. According to a recent study ^[46] on a leporine model of long bone defects, the therapeutic potential of autologous BMA clot in the repair of ulnar defects was higher than that of autologous bone grafts, since it was associated with a lower risk of complications. Bone graft fragments (the current main strategy in the use of bone graft augmentation surgeries) may prolong the regenerative cascade, since the body needs to process and resorb microparticles. This is not the case with autologous clot.

Although the therapeutic strategies for the use of BMA clot may vary, most of the studies are intended for the enhancement of cartilage and bone regeneration, being predominantly of preclinical classification. Taking the aforementioned investigations into consideration, the biological potential of bone marrow aspirate products is further illustrated as a beneficial ally in regenerative medicine.

5. Fibrinolytic Mechanisms

It is known that platelets are also found in bone marrow products and carry a milieu of growth factors and proteins (Table 1). These molecules are strongly involved in the fibrinolytic system and may upregulate or downregulate fibrinolytic reactions. The temporal relationship and relative contributions of hematological components (as well as the function of platelets in clot degradation mechanisms) are still a matter of great discussion ^[47]. The literature presents many investigations thoroughly discussing the role of specific cell populations, proteins, cytokines and platelets, which have long been known for their ability to enhance the healing process. Despite numerous studies, additional hematological components (such as coagulation factors and the fibrinolytic system itself) have also been found to be of equal importance when it comes to wound repair ^[42]. Fibrinolysis is a complex biological process which relies on the activation of specific enzymes in order to promote degradation of fibrin ^[42]. Fibrinolytic reactions have been previously discussed by researchers ^[48], who suggested that fibrin degradation products (FDPs) may actually be responsible for driving tissue repair, preceding the sequence of biological events from fibrin deposition and removal all the way to angiogenesis (which is essential for wound healing).

Regulated by plasmin, the fibrinolytic system exerts key roles in promoting cell migration, growth factor bioavailability, and the regulation of other protease systems involved in tissue inflammation and regeneration ^[49][50]. The main components present in fibrinolytic reactions, e.g., urokinase plasminogen activator receptor (uPAR) and plasminogen activator inhibitor-1 (PAI-1), are present in MSCs, the specialized cell types necessary for successful wound healing ^[49].

These cells are known to be mobilized from the bone marrow under adverse conditions (such as severe organ injury) and may therefore be detected in the circulation of individuals with multiple bone fractures, for instance. On the other hand, under specific conditions such as end-stage kidney and liver failure, or during rejection episodes following heart transplantation, these cells may not be detected in blood ^[51]. It is interesting to note, however, that human bone marrow-derived MSCs may not be detected in the blood of healthy individuals ^[52].

A hypothesis for MSC mobilization has previously been put forward by uPAR, and remains similar to the mobilization process of another cell type present in the bone marrow, the hematopoietic stem cell (HSC). One in vivo study ^[53] revealed that the urokinase receptor controls mobilization, migration, and differentiation of MSCs, indicating a supportive role for the fibrinolytic system in regenerative processes. Vallabhaneni et al. observed that uPAR was required for MSC mobilization from the BM of mice who were stimulated with granulocyte colony-stimulating factor (G-CSF), as an insignificant amount of these cells was mobilized in uPAR deficient mice when compared to the eight-fold increase in wild types ^[53]. Furthermore, uPAR-deficient mice demonstrate upregulated expression of G-CSF and stromal cell-derived factor 1 (CXCR4) receptors in BM; the downregulation of uPAR inhibits migration of MSCs, including the ones of human origin. Lastly, the authors concluded that either down- or upregulation of the urokinase receptor can inhibit or stimulate MSC differentiation, respectively ^[53]. Others have shown that the glycosylphosphatidylinositol-anchored uPA receptor can control adhesion, migration, proliferation, and differentiation events. This can be achieved through the activation of certain intracellular signaling pathways, such as the prosurvival phosphatidylinositol4,5-bisphosphate 3-kinase/Akt and ERK1/2 signaling, as well as the focal adhesion kinase (FAK) ^[54][55].

6. The Role of Hyaluronic Acid in the BMA Matrix

Hyaluronic acid (HA) or hyaluronan is an anionic, nonsulfated glycosaminoglycan that is widely distributed throughout connective, epithelial, and neural tissue ^[56]. Hyaluronan is also an important component of articular cartilage in which it exists as a coating surrounding each chondrocyte. It carries lubricant properties for tendons and joints, and reduces the breakdown of extracellular matrix by inhibiting metalloproteinase synthesis. Its anti-inflammatory properties downregulate the activity of tumor necrosis factor-alpha and interleukin-1, two major inflammatory aggressors.

In a respectable study, authors evaluated the use of fibrin and hyaluronic acid mixtures as an ally in the regenerative potential of bone marrow concentrates ^[57]. Histologically, the bone marrow concentrate and hyaluronic acid plus fibrin groups exhibited significant chondral regeneration, with faster and more complete healing of the osteochondral hole in the trochlear region of rabbit knees (in comparison to the hyaluronic acid plus fibrin only group and controls). Furthermore, scanning electron microscopy images of hyaluronic acid/fibrin composites mixed with bone marrow cells revealed that mixing fibrin with hyaluronic acid enabled it to polymerize from solid to porous structures through which cells attached and could be stimulated to proliferate.

Hyaluronic acid and fibrin mixtures are supposed to have a fundamental role in creating an effective connection between cells (Figure 1). In this sense, HA might influence the polymerization process of fibrin turning it into a more spongy structure that would otherwise be formed by the solely natural set of fibrinogen and thrombin. This arrangement seems to be critical for cell adherence, both locally and peripherally.

From a safety standpoint, fibrin sealants are biological adhesives that mimic the final step of the coagulation cascade; therefore, an association with hyaluronic acid injection may be considered a biologically safe and natural approach ^[58].

In a gout model study, articular cartilage regeneration with autologous bone marrow aspirate and hyaluronic acid was demonstrated in detail ^[59]. Interestingly, full-thickness cartilage defects were created and animals were given either no injections or weekly sessions of HA or HA plus BMA for three weeks. The addition of HA alone managed to improve the quality of repaired tissue (e.g., hyaline-like cartilage). However, the combination of HA plus BMA yielded the best results with vertical orientation of chondrocyte nests, and the presence of type II collagen and proteoglycans in the intermediate and deep cartilage layer (more safranin O staining than the other two groups); and type I collagen confined to the superficial layer and perichondrium. Chondrocyte hypertrophy was not observed. Authors concluded that after arthroscopic subchondral drilling, the technique may optimize articular cartilage repair.

In a randomized controlled trial, articular cartilage regeneration with autologous peripheral blood stem cells (PBSC) combined with HA versus HA alone was studied ^[60]. Fifty patients with ICRS (International Cartilage Repair Society) grade 3 and 4 lesions of the joint were allocated to either receive a series of weekly injections either HA or PBSC + HA. Subjective IKDC (International Knee Documentation Committee) scores and MRI (magnetic resonance imaging) scans were obtained preoperatively and postoperatively at serial visits. Additionally, a second-look arthroscopy and biopsy were performed to grade the regenerated tissue through ICRS II (International Cartilage Repair Society Visual Assessment Scale II). On ICRS II histologic and morphologic MRI scores, the combined injections yielded statistically better results reflecting the quality of cartilage repair, which was represented by the sustained clinical improvement at 24 months in the PBSC + HA group.

Previous contributions have been made, in which the effect of HA mixed with PRP (the PRP matrix) showed better clinical outcomes (Western Ontario and McMaster Universities Arthritis Index (WOMAC) and Visual Analogue Scale (VAS)) over HA or PRP alone ^[61]. The authors concluded that the lubrication and support to the extracellular matrix that HA provided seemed to enable earlier functional benefits to the PRP injection. This may ameliorate rehabilitation and anticipate a patient’s return to routine activities. Considering these observations, we argue that the BMA Matrix may share similar mechanisms and effects—which have been previously suggested in the numerous studies cited in this manuscript.