Biologics in Orthopaedic Surgery

The Importance of Concentrating Bone Marrow Aspirate

Without concentration techniques, bone marrow aspirate alone has a low percentage of MSCs of only 0.001%–0.01% of nucleated cells. For this reason, centrifugation is used to concentrate bone marrow aspirate in order to increase the percentage of MSCs. In bone marrow aspirate, the predominant cell type is neutrophils and erythrocytes. With this in mind, there have been gender differences noted in the predominant cell type among bone marrow aspirate with male aspirate containing more erythroblasts and female aspirate containing more neutrophils. In general, the cellular composition of BMAC is 28.1% erythroblasts, 32.7% neutrophils, 13% lymphocytes, 2.2% eosinophils, 1.3% monocytes, and 0.1% basophils. In comparison to platelet-rich plasma (PRP), the cellular composition of BMAC has 11.8 times the amount of white blood cells, 19.4 times the amount of neutrophils, and 2.5 times the amount of platelets. Similar to PRP, BMAC contains a comparable concentration of monocytes, lymphocytes, eosinophils, and basophils.

The importance of concentrating bone marrow aspirate into BMAC has been highlighted by several studies supporting a direct correlation of the number of cells with healing potential and optimal outcomes. With this in mind, the minimum number needed, to induce biologic healing, is 1000 progenitors/cm ³ of BMAC. In like manner, a study on bone healing of nonunions showed a statistically significant lower rate of union in patients who received a BMAC with less than 1000 progenitors/cm ³ and less than 30,000 progenitor cells in total. At the same time, this study found a positive correlation between the number and concentration of osteoprogenitor cells delivered in the bone marrow aspirate with the volume of mineralized callus present at the sites of bone healing. All things considered, Hernigou et al. concluded that 1 mL of bone marrow aspirate contained a competent concentration of cells to form 1 cm of bone in areas of nonunion. Further emphasis has been placed on the critical concentration of certain cellular elements, such as MSCs, rather than the total cell count of the entire composition of cells contained within BMAC. As can be seen, the concentration of bone marrow aspirate is imperative to obtain the optimal volume of the essential cellular elements, i.e., MSCs, recommended to expedite the biologic healing response; yet, BMAC also provides a source of growth factor therapy to enhance treatment.

Growth Factors and Cytokines Contained in Bone Marrow Aspirate Concentrate

Growth factors are biologically active polypeptides that can be applied to stimulate cell growth and enhance both chondrogenesis and osteogenesis. Commonly encountered growth factors, pertinent to healing of musculoskeletal tissue, are transforming growth factor-β2 (TGF-β2), PDGF, fibroblast growth factors (FGFs), VEGF, interleukins (ILs), Insulin-like growth factor-1 (IGF-1), and BMPs ( Table 4.1 ). BMAC, not to mention, has an increased concentration of growth factors due to the substantial number of platelets with α-granules. Specifically, the α-granules of platelets contain TGF-β2, PDGF, VEGF, FGF, BMP, and IGF. In comparison to PRP, BMAC has a significantly increased supply of growth factors with 172.5 times more VEGF, 78 times more IL-8, 4.6 times more IL-1B, 3.4 times more TGF-β2, and 1.3 times more PDGF. Each of these aforementioned growth factors has specific functions to stimulate healing in select tissues important in orthopaedics. As an illustration, the most common growth factor used to stimulate chondrogenesis is TGF-β2, which stimulates extracellular matrix synthesis and chondrogenesis in the synovial lining all while decreasing the catabolic activity of IL-1. Explicitly, the proliferation and differentiation of chondrocytes by TGF-β2 is via phosphorylation of SMAD (Suppressor of Mothers Against Decapentaplegic) proteins that transduce extracellular signals to the nucleus to active downstream gene transcription for chondrogenesis. In the same fashion, BMPs have been shown to stimulate, through autocrine signaling, both osteogenesis and chondrogenesis. In particular, BMP-2 has been used to stimulate bone growth in the setting of fracture healing, while BMP-7 has been used to stimulate cartilage matrix synthesis by inhibiting catabolic factors of matrix metalloproteinases. ILs, involved in the initial inflammatory response to injury, aid in cell migration to the site of injury while increasing production of other favorable factors, such as VEGF. In the same fashion, VEGF promotes angiogenesis to provide transport of necessary products for healing and growth of mesenchymal tissue. Further healing is augmented with PDGF as a chemotactic factor for mesenchymal cells and suppressor of IL-1β, which induces cartilage degradation by downregulation of nuclear factor-κβ. PDGF, at the same time, stimulates wound healing and collagen synthesis and promotes formation of cartilage. Another growth factor, commonly contained in BMAC, is IGF-1 that increases collagen and proteoglycan synthesis, increases metabolic activity, and helps maintain the integrity of articular cartilage. A large family of growth factors is FGFs with special emphasis for FGF-2 and FGF-18, both of which are involved in chemotaxis of MSCs and chondrogenic differentiation. Taken together, the growth factors and stem cells contained in BMAC are individually important in augmenting the biologic healing of musculoskeletal tissue.

Sources of Bone Marrow Aspirate

The aspiration of bone marrow can be performed at various locations throughout the human body. Despite the different anatomic locations available for bone marrow aspiration, all sites of aspiration do not provide the same proportion of BMAC. In fact, the quantity of progenitor cells, from bone marrow aspirate, decreases from axial to appendicular skeleton as well as from proximal to distal in the appendicular skeleton. The highest quantity of progenitor cells in bone marrow aspirate has been found in the vertebral bodies. Although this may be true, aspiration of bone marrow from the vertebral body is not as practical as other available anatomic sites for aspiration. More reasonable locations for aspiration have been obtained from bone near the site of surgery or the iliac crest, which is based on both accessibility and percentage yield of progenitor cells. In like manner, a study comparing sites to harvest BMAC from the iliac crest, tibia, and calcaneus found that the highest concentration of progenitor cells resided in the iliac crest. Equally important, this study demonstrated that all anatomic sites of harvest, even the proximal tibia and calcaneus, are both safe and amenable to achieve adequate amounts of progenitor cells. By the same token, no difference in cell yield was noted with demographics of age or sex or with comorbidities of smoking and diabetes. Given the accessibility of the iliac crest, this anatomic site is one of the most commonly used locations for obtaining bone marrow aspirate. For this reason, Pierini and colleagues further evaluated the yield of progenitor cells from either the anterior or posterior iliac crest. In comparison to both sites of the iliac crest, the highest yield of colony-founding connective tissue progenitor cells was aspirated from the posterior iliac crest. However, no difference in the biologic potential in terms of viability or differentiation of MSCs was noted between the anterior or posterior iliac crest. In spite of this study, another study by Marx and colleagues found no significant difference in the yield of either stromal stem cells or hematopoietic stem cells between the anterior or posterior iliac crest. Rather, both the anterior and posterior iliac crest harvest provided twice the amount of stromal and hematopoietic stem cells than harvest from the tibial plateau. As can be seen, the iliac crest is the preferred anatomic location for aspiration of bone marrow, yet other anatomic sites, near the area of surgery, may be able to provide the principal BMAC preparation required for healing.

The Bone Marrow Aspirate Concentrate Technique

The technique of harvesting BMAC can be applied to most anatomic locations. For simplicity, the technique will be further described in reference to the iliac crest, which is the most commonly used site for aspiration of bone marrow. The technique begins with a percutaneous stab incision placed over the iliac crest. Without delay, a Jamshidi needle is directed parallel to the iliac wing between the inner and outer tables of the crest. With use of the Jamshidi needle two starting holes are made, with one hole used to collect the first 30 mL of aspirate and the second hole used to collect the second 30 mL of aspirate. To begin with, 5 mL of marrow is aspirated, and then the Jamshidi needle is rotated 45 degrees to begin the next aspiration. After the next 5 mL of bone marrow is aspirated, the Jamshidi needle is advanced 1–2 cm, and then the process is repeated with another 5 mL aspiration followed by a 45-degree rotation of the needle. With each advancement and reposition of the needle, the obturator is reinserted into the needle in order to clear the bore of the needle from debris. Importantly, the vacuum aspiration of bone marrow occurs in conjunction with anticoagulation via heparin/ACD-A solution (8 mL of anticoagulant per 60 mL of bone marrow aspirate). In due time, with aspiration of the desired volume of bone marrow, the fluid is concentrated with use of a centrifuge system. Straightaway processing and concentration of bone marrow aspirate takes place with density gradient centrifugation using an automatic microprocessor-controlled centrifuge system. Following about 15 minutes of centrifugation, the erythrocytes, nucleated cells, and plasma are separated into a second chamber. Completion of the centrifugation process yields approximately 7–10 mL of BMAC available for injection into the designated area to augment biologic healing.

Several studies have investigated multiple methods to optimize the technique of harvesting bone marrow aspirate. These studies have evaluated the needle advancement, the syringe size, the aspirate volume, and the different commercial systems available for BMAC harvest ( Table 4.2 ). With attention to needle placement, movement of the needle throughout the aspiration process provides a higher yield than a stationary needle. Peters and colleagues demonstrated that multiple needle advancements resulted in a higher concentration of MSCs. In particular, needle advancement of 5 mm up to 3 times throughout the procedure increases the proportion of MSCs. Not to mention, the size of the syringe used for vacuum aspiration of the bone marrow matters in regard to cell yield. In a study by Hernigou et al., a 10 and 50 mL syringe were compared during bilateral aspiration of bone marrow from the iliac crest. In comparison, there was a 300% higher cell yield with a 10 ml syringe, and equally important, there were significantly more cells in the first 1 mL aspirate of the 10 mL syringe as opposed to the first 5 mL of the 50 mL syringe. As a result, the more efficient technique to optimize aspiration of bone marrow is by aspirating smaller volumes in small syringes. By the same token, larger volumes of aspirate appear to dilute the concentration of bone marrow. As small a difference as 2 mL of bone marrow aspirate can produce a drastic difference in the amount of MSCs. Indeed, Muschler and colleagues demonstrated a difference between 2 and 4 mL of bone marrow aspirate from the anterior iliac crest of patients with the number of MSCs decreasing by 50% from the higher volume of aspirate. In like manner, a study by Bacigalup et al. determined that harvest of bone marrow using multiple small volume aspirations decreased the dilution with peripheral blood, thus resulting in a higher yield of MSCs. Multiple commercial companies provide systems for harvesting of bone marrow aspirate. A study comparing different systems to harvest BMAC included the following systems: Harvest SmartPRep2, Biomet BioCUE, and Arteriocyte Magellan. In comparison of these systems, the Harvest system showed significantly greater concentration of connective tissue progenitors both before and after centrifugation in comparison to the Biomet BioCUE system. In a similar fashion, the Harvest system showed significantly higher percent yield of connective tissue progenitor cells after centrifugation in comparison to the Arteriocyte Magellan system. Under these circumstances, the difference between systems, in harvest of bone marrow aspirate, is believed to be due to differences in the centrifugation device that leads to a variation in yield of concentrated progenitor cells.

The Application of Bone Marrow Aspirate Concentrate

The utility of BMAC has been applied to address numerous pathologies of the musculoskeletal system. As an illustration, BMAC has been used to induce and increase bone formation. In an analysis of basic science evidence for long bone healing, Gianakos et al., summarized that several studies support BMAC significantly increasing the amount of bone formation. To demonstrate this finding, imaging showed significant increases in bone formation by an increase in bone volume, callus formation, and union of healing bone. At the same time, on microcomputed tomography of animal subjects receiving BMAC, there was an 81% reported significant increase in the bone area of these study subjects. Under closer observation, histologic and histomorphometric assessment of BMAC subjects showed a 90% significant improvement in earlier bone healing. The increased bone formation, in bone defects treated with BMAC, translated into a higher torsional stiffness of 78%. In like manner, BMAC, in addition to adding bone, has also exhibited the ability to add cartilage and augment cartilage healing. Must be remembered, BMAC contains MSCs that have been validated to increase aggrecan content and tissue firmness that together compliment the cartilage repair. In a goat model by Saw and colleagues, surgically created cartilage defects when treated with BMAC, along with hyaluronic acid (HA), promoted the healing of cartilage with more glycosaminoglycan and a higher content and a better organization of hyaline cartilage. In an equine model by Fortier and colleagues, cartilage defects were treated with either microfracture alone or microfracture along with BMAC. In comparison of both groups, the addition of BMAC resulted in increased glycosaminoglycan, increased type II collagen, improved collagen orientation, and improved filling of cartilage defects. Important to realize, the remarkable results in animal models has translated into optimized outcomes in human patients. In a study by Gobbi et al., BMAC was compared with matrix-induced autologous chondrocyte implantation (MACI) for treatment of patellofemoral chondral lesions. Although both groups of patients reported significant improvements, patients treated with BMAC showed significantly greater improvements in functional outcome scores. Summation of clinical studies of BMAC, through a systematic review by Chahla et al., has synthesized good to excellent overall outcomes with treatment of BMAC for both knee osteoarthritis and knee chondral defects. Another key point, from this systematic review, was that BMAC has better outcomes for treatment of cartilage lesions in patients younger than 45 years, with smaller size of chondral lesions, and with a fewer number of lesions. Even though there is a broad range of clinical applications for BMAC from healing of fractures to treatment of osteoarthritis or cartilage defects, future studies are warranted to further elucidate the exact mechanisms and particular patients that would most benefit from augmentation of biologic healing with BMAC.

Safety of Bone Marrow Aspirate Concentrate

As with all treatment inventions, the safety and risk of potential complications must be weighed against the benefits of the BMAC procedure. The use of BMAC requires harvesting the bone marrow from a donor site of the patient; this entails an additional area of interruption and potential increased morbidity from the surgical technique. For fear of morbidity, the safety of autologous BMAC was evaluated by Hendrich et al., in the initial experiences in 101 patients. After an average of 14 months, upon reexamination of all the patients, no major morbidity or mortality was directly due to the BMAC treatment. In particular, there were no noted infections, no induction of tumor formation, and no noted complications at the harvest site of the iliac crest. Given the contents of BMAC containing stem cells, there is concern of cancerous formation from these pluripotent cells. With this in mind, the risk of cancer was explored by Hernigou et al., in patients treated for orthopaedic diseases with autologous bone marrow cell concentrate. At a mean of 12.5 years of follow-up, there was 1873 patients treated with BMAC, and after reviews of more than 7300 magnetic resonance images (MRIs) and 52,000 radiographs, there were only 53 cancers diagnosed and not one of these cancers occurred in areas of the body that received an injection of BMAC. On a less severe scale of complications, one of the most common adverse events reported, after BMAC treatment, were swelling and pain at either the harvest site or the site of injection. As an illustration, these common complications of swelling and pain were noted in the majority of the 75 degenerative knees that received BMAC for osteoarthritis. On the contrary, in another study by Centeno and colleagues, after 840 BMAC procedures for knee osteoarthritis, no severe adverse events post procedure were due directly from the BMAC technique. In detail, symptoms after BMAC treatment were 6% for those patients who only received BMAC and 8.9% for those patients who received BMAC with adipose graft with the majority of these symptoms being mostly self-limited pain and swelling. As a result, the consensus of these studies conclude that BMAC treatment has no significant increase risk of cancer, limited harvest site morbidity, and the most common complications being self-limited symptoms without severe consequence.