Mechanism of Action and Clinical Applications

Currently, there are no guidelines on PRP dosing and the use of specific formulations for different pathologies. However, some studies have reported LP-PRP may be beneficial for cartilaginous injuries and LR-PRP for tendinous injuries. However, other studies report that LR-PRP should be avoided in osteoarthritis and tendinopathies because WBCs release proteases and reactive oxygen species that may harm remaining cartilage or tendinous tissue, respectively. One study noted that compared to treatment with LP-PRP and phosphate-buffered saline (PBS), synovial cells treated with LR-PRP resulted in significantly greater cell death and some proinflammatory mediator production. While the exact dosing and preparation are unknown, some studies have recommended that the platelet concentration for knee osteoarthritis and tendinopathies should target close to 3 to 4× and be leukocyte free. ^,

There is no clear in vivo mechanism for the therapeutic effects of PRP and its subtypes; however, PRP’s therapeutic effects are often attributed to bioactive factors and high concentration of growth factors. The true biologic response to PRP is poorly understood and likely pleiotropic, and can have positive benefits on one tissue and deleterious effects on other. In vitro, platelets play important roles in wound healing including re-epithelialization, granulation tissue formation, cartilage and bone matrix formation/remodeling, chemotaxis, and angiogenesis. ^, Platelets accomplish this through their alpha granules that contain the necessary growth factors for these processes, such as platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), and fibroblast growth factor-2 (FGF-2) ( Table 7.1 ). They also produce tissue growth factor-β1 (TGF-β1)—one of the most important mediators in connective tissue regeneration and potent inhibitors of matrix metalloproteinases (MMP-1, -3, -9), which inhibit collagen synthesis.

It is proposed that growth factors from PRP induce processes of inflammation and cell recruitment, angiogenesis, and matrix formation by delivering a large quantity of platelets to the site of injury; however, this is still the subject of ongoing research. It is important to note that PRP’s effects through a complex sequence of growth factor communication within the tissue microenvironment facilitates healing of the remaining tissue and does not regenerate absent tissue.

Common clinical applications for PRP use include, but are not limited to: tendinopathies (patellar, rotator cuff, gluteal, lateral epicondylitis, plantar fasciitis), ligamentous tears (ulnar collateral ligament, anterior cruciate ligament [ACL]), muscular strains (hamstring, gastrocnemius, lumbar multifidus), peripheral mononeuropathies (carpal tunnel syndrome), spine pathology (sacroiliitis, facet arthropathy, degenerative disc disease, and radiculopathy), and peripheral joints (knee osteoarthritis, carpometacarpal joint pain, and temporomandibular joint syndrome).

Data on Efficacy

The main limitation for clinical use of PRP is not a lack of data but rather heterogeneous data. There is a large variation in standardization in its preparation and delivery, making it difficult to discern potential beneficial effects of PRP. For instance, while PRP has demonstrated overall favorable outcomes in some systematic reviews, the studies had varying methods of PRP preparation, patient populations, and stages of disease.

Shen and colleagues conducted a systematic review that found that most patients had significantly improved pain relief and self-reported function after PRP injection for knee osteoarthritis compared to saline placebos, hyaluronic acid (HA), ozone, and corticosteroid injections. It was also reported that 10 of the 14 included studies had high risk of bias associated with them, mostly due to lack of investigator blinding. Laver and colleagues published a recent systematic review of PRP in knee and hip osteoarthritis encompassing 29 studies, including 1 hip and 8 knee randomized controlled trials (RCTs) and 11 total studies controlled with HA. For the studies controlled with HA, 9 of 11 studies found that PRP was significantly superior to HA in terms of symptom improvement. In general, beneficial effects of PRP therapy in osteoarthritis have been more effective in cohorts with milder disease and younger populations. ^,

A 2017 systematic review and meta-analysis comprised exclusively from Level 1 RCTs in rotator cuff injuries and lateral epicondylitis reports significantly less pain in PRP-treated groups compared to control in the long term. This differs from prior meta-analysis, largely including low-powered studies and heterogenicity in comparator outcomes, which have published contrasting results.

A double-blinded RCT by Dragoo and colleagues compared the clinical outcomes in patients with patellar tendinopathy after a single ultrasound-guided, leukocyte-rich PRP injection with dry needling compared to dry needling in 23 patients. They reported that while the regimen of standardized eccentric exercise and PRP injection with dry needling appeared to help patients with patellar tendinopathy more than dry needling alone at 12 weeks ( P = .02) as evaluated by the Victorian Institute of Sports Assessment (VISA), the benefits of PRP was not long lasting ≥26 weeks ( P = .66). An RCT of a single injection of LR-PRP or LP-PRP was not found to be more effective than saline for the improvement of patellar tendinopathy symptoms in a study by Scott and colleagues. There was no significant difference in the VISA score, pain during activity, or global rating of change at 12 weeks, 6 months, and 12 months. A systematic review and meta-analysis of nonsurgical treatments of patellar tendinopathy by Andriolo and colleagues noted that while eccentric exercises seem to be the most beneficial with short-term follow-ups less than 6 months ( P < .05), multiple PRP injections may offer more satisfactory results at long-term follow-up (≥6 months, P < .05).

Wu and colleagues conducted a single-blind RCT to assess the 6-month effect of PRP in patients with mild to moderate carpal tunnel syndrome (CTS). Sixty patients with unilateral CTS were randomized into two groups of 30, with one group receiving one dose of 3mL of PRP using ultrasound guidance and the control group receiving a night splint. The patients who received PRP exhibited a significant reduction in the visual analog scale (VAS), Boston Carpal Tunnel Syndrome Questionnaire score, and cross-sectional area of the median compared to those of the control group 6 months post-treatment ( P < .05).

A systematic review and meta-analysis by Sheth and colleagues comparing PRP to control therapies (such as physiotherapy or placebo injections) in acute muscle injuries (≤7 days) found that while the use of PRP did not significantly decrease the rate of injury at 6 months follow-up, there was significant decrease in return to play times except with a subgroup with grade 1 or 2 hamstring muscle strain. A literature review by Setayesh and colleagues on the treatment of muscle injuries with PRP found that while there were numerous clinical case series demonstrating faster healing, less swelling, and quicker return to play times for patients with muscles strains who received PRP, most studies were retrospective and few RCTs demonstrate a clear clinical benefit. They also observed variability in the injectant preparation, the platelet concentration, the presence of leukocytes, the volume of PRP injected, and the timing of treatments.

Figueroa and colleagues conducted a systematic review of prospective cohort studies or RCTs on PRP compared to control in the treatment of ACL tears, assessing graft-to-bone healing, graft maturation, and/or clinical outcomes. While they found that more studies had evidence of faster graft maturation, it appears there were mixed results, with overall findings leaning to no significant improvement in graft-to-bone healing or clinical outcomes.

Overall, there is currently a need for systematic characterization of PRP therapy, which along with appropriately conducted prospective trials, could better understand PRP’s efficacy and formulations.

Caveats/Side Effects

PRP has demonstrated an excellent safety profile with no reported major adverse events or reactions. The side effects are similar to those for any intra- or extraarticular injection, such as infection, pain, bleeding, and injury to nearby blood vessels or nerves.

Drug interactions with PRP have not been well studied and documented in literature. Nonsteroidal antiinflammatory drugs (NSAIDs), such as aspirin and naproxen, have been shown to interfere with growth factor release given the direct inhibition of cyclooxygenase pathway. ^, Although there is no research correlating growth factor release and efficacy of PRP treatment in vivo, these studies suggest NSAIDs and aspirin should be held prior to PRP, and other analgesics, such as acetaminophen, should be prescribed for post-injection pain control.

US Food and Drug Administration (FDA) Regulations

PRP is regulated under the FDA Center for Biologics Evaluation and Research (CBER), which exempts it from the traditional regulatory pathway. The 510(k) application for “substantially equivalent” devices is used to bring PRP products to market. There are a myriad of FDA-approved devices ranging from traditional, injectable PRP to PRP-based biomaterials intended for enhancing bone graft healing. Most of the 33 systems on the market have FDA 510(k) clearance to enhance bone graft preparations with PRP. Although PRP injections are considered off-label use, physicians are allowed to administer them under CBER as long as they are “well informed about the product…base its use on firm scientific rationale and on sound medical evidence, and… maintain records of the product’s use and effects.”

The World Anti-Doping Agency (WADA), an international independent agency that regulates doping-free sport, had prohibited PRP in 2010, but removed it from the list in 2011. The initial concern from the experts was that growth factors in PRP may stimulate muscle growth beyond the normal physiologic state and give individuals an unfair advantage; however, the prohibition was lifted as there was limited evidence for a systemic ergogenic effect of PRP. PRP appears to trigger an increase in circulating growth factors systemically, including elevated serum insulin-like growth factor-1 (IGF-1), VEGF, and basic fibroblast growth factor (bFGF), which may be banned by governing agencies, and should be used with caution in athletes as this may cause false negatives in doping tests.

Conclusion

PRP continues to be a promising therapy, especially in early osteoarthritis- and tendinopathy-related injuries. However, its use is currently limited by mechanistic understanding, cost burden, a lack of standardization in its formulation protocols, and calls for more robust blinded studies.

Mechanism of Action and Clinical Applications

The mechanism of action of PL is poorly understood; however, theoretically, the mechanism of action and clinical effectiveness should be similar to PRP. There are no clear indications for the use of PL, but case reports and case series have reported the use of PL for knee osteoarthritis, lumbar radiculopathy, and lateral elbow tendinopathy. ^{, ,} It has been theorized that PL may be of more benefit around nerves as it is thought to be more antiinflammatory than PRP. However, this has not been studied in detail and there is no published literature on the effect of PL on nerve injuries outside of the Centeno and colleagues’ lumbar radiculopathy study mentioned above.

Data on Efficacy

There are limited data regarding clinical efficacy of PL. In a prospective open-label study of 48 patients, Al-Ajlouni and colleagues suggests intra-articular (IA) injection of autologous PL in patients with osteoarthritis of the knee is effective in reducing pain and restoring function without provoking local or systemic adverse events. This is consistent with the in vitro study in 2017 that suggested PL induces quiescent cartilage cell activation and proliferation leading to new cartilage formation. In 2017 Centeno and colleagues suggested in a retrospective review of 470 patients that PL decreases pain in lumbar radiculopathy with a significantly improvement in pain scores up to 2 years post-injection. Tan and colleagues observed in a prospective study of 56 patients significant improvement in VAS score and Mayo score for function following PL in chronic lateral epicondylitis who failed conservative therapy. There are limited data regarding clinical efficacy of PL, or in vivo study of PL on human cells.

Caveats/Side Effects

Studies on clinical efficacy of PL are limited to small case series or case reports, and the study results have not been repeated. PL as an autologous therapy has similar complications and side effects to PRP. A more comprehensive review of drugs and their effects of PL remains to be conducted, but it is possible aspirin and NSAIDs can cause an inhibitory effect on growth factor release, and therefore should be withheld in the days prior and after injection.

FDA Regulations

The use of PL is not FDA approved, however, its off-label use may be allowed by regulatory exemption per Title 21 United States Code of Federal Regulations Part 1271 (21 CFR 1271) and the 361-product exemption. In the author’s opinion, PL is exempt as it is derived from an autologous source, has a homologous use of tissue, and is processed with minimal manipulation.

Mechanism of Action and Clinical Applications

In the IA environment, A2M inhibits endoproteases, particularly matrix metalloproteinases, responsible for catabolic activity. Although A2M is found in the serum, A2M is a large molecule and does not cross into synovial fluid and joint spaces at large concentrations for significant IA effect. Injection of autologous A2M concentrate, such as Cytonics APIC System, into the joint allows for the in vivo delivery of high-volume A2M, which can bind and inhibit catabolic enzymes, inhibit the inflammatory cascade, and create a favorable healing environment in the joint or other area of tissue pathology.

Data on Efficacy

Although used clinically, A2M has not been extensively studied and to date there is minimal in vivo human studies. Of the available studies, most have involved assessing osteoarthritis of the knee in preclinical models. Elevated levels of catabolic proteases and cytokines in synovial fluid have been shown to induce chondrocyte death and cartilage matrix degeneration. In a rat model of osteoarthritis induced by ACL transection, IA A2M provided chondral protection and treated knees demonstrated less cartilage damage compared to controls. ^, There is also suggestion in a rabbit model that it may help in healing of a ruptured ACL. Theoretically, since A2M is thought to be antiinflammatory, it is thought to potentially have utility in patients with increased joint inflammation or inflammatory arthropathies. An in vivo study by Cuellar suggests there may be efficacious use of A2M in discogenic pain in patients positive for the fibronectin-aggrecan complex (a protein biomarker of disc disease), as measured by VAS and Oswestry Disability Index (ODI) scores at 6 months following an intradiscal injection.

Caveats/Side Effects

There are limited data on clinical efficacy and there are no known specific risks of A2M other than general adverse procedural risks.

FDA Regulations

A2M is not FDA approved; however, its off-label use may be allowed by regulatory exemption per 21 CFR 1271 and the 361-product exemption. In the author’s opinion, current products to concentrate A2M from whole blood undergo “minimal manipulation” and therefore would be exempt.

Mechanism of Action and Clinical Applications

The proposed mechanism of action of IRAP is by competitive inhibition at the IL-1 receptor, blocking the pro-inflammatory IL-1 effect. There are no clear indications for the use of autologous IL-1Ra at this time. Some possible indications are in osteoarthritis of major joints. In osteoarthritis specifically, IL-1 directly contributes to cartilage loss through upregulation of extracellular proteolytic enzymes while simultaneously suppressing cartilage anabolism through downregulation of collagen and proteoglycan synthesis. ^,

Data on Efficacy

A 2019 systematic review of IL-1Ra products for knee osteoarthritis concluded that autologous IL-1Ra may improve pain and functionality for mild to moderate OA, and may be an effective adjunct for those unresponsive to traditional IA therapies. In a randomized, double-blind, placebo-controlled clinical trial in knee OA patients a one-time injection of IL-1Ra significantly improved pain scores compared to placebo; however, there was no significant difference 4 weeks after the injection. ACS with high-concentration IL-1Ra has also been studied for the treatment of lumbar and cervical radiculopathy with improvement in pain, function, and quality of life similar to that of corticosteroids with epidural injections, but with possibly a longer efficacy. There is early preclinical work in a rat model of tendon disease, where IL1-Ra improved structural endpoints with histologic analysis.

Current research is exploring long-term IL-1Ra delivery by gene therapy, or injecting the gene encoding the protein using a virus vector. This has been proposed to be a superior method of increasing IA IL-1Ra expression. Gene therapy IL-1Ra has been studied in animal models and found to be safe, symptomatically effective, and disease modifying; however, to date, there are no human trials.

FDA Regulations

There is no FDA-approved system to produce autologous whole-blood–derived IL-1Ra proteins at this time. It is the opinion of the author that the Orthokine system (or Regenokine in the United States) to produce ACS is more than minimally manipulating the whole blood product, and thus would not categorize under regulatory exemption per 21 CFR 1271 and the 361-product exemption.

Mechanism of Action and Clinical Applications

While the exact mechanism of how BMAC functions is not known, the various components of BMAC have been shown to induce differentiation and proliferation of resident stem cells, and have chondrogenic and osteogenic potential, as well as antiinflammatory effects in vitro. HCSs, also found in BMAC, can differentiate into red blood cells and muscle, are involved in muscle repair, and stimulate osteogenesis with direct contact with MSCs. ^, In addition to the platelet’s ability to release growth factors to initiate stem cell migration to the injury site and provide adhesion sites for migrating stem cells, platelets can also decrease pain via a peripheral endocannabinoid related pathway. ^,

There is evidence that the dose of progenitor cells can affect clinical outcomes. Hernigou reported that for the treatment of nonunion tibia fractures with BMC, the total number and concentration of bone marrow progenitor cells had a significant effect on fracture healing. Pettine reported that in the treatment of discogenic pain with BMC, higher MSC concentration was linked to pain reduction post procedure. Centeno reported that in the treatment of knee arthritis with BMC, in patients with a total nucleated cell (TNC) count above 400 million there was significant improvement in all studied pain and functional metrics compared to the lower cell count group. Both groups did report significant improvement from baseline.

The addition of a supplementary PRP injection to the BMAC treatment protocol can potentially influence the results of an isolated BMAC injection for OA. A single, image-guided IA injection of BMAC followed by a supplementary IA injection with PRP at 8 weeks follow-up was well-tolerated and significantly improved pain at short-term follow-up among patients with moderate-to-severe OA. An ongoing clinical trial called the CASCADE trial is examining the use of BMAC with and without HA for discogenic low back pain. ^,

Data on Efficacy

Few studies have compared the use of noncultured (nonexpanded) BMSCs with cultured (expanded) stem cell therapy, and it is unclear which method may be superior. While there are no RCTs on the efficacy of noncultured BMAC for patients with cartilage disease, there have been many publications illustrating its safety profile and therapeutic potential. Furthermore, the harvested stem cell quantity and quality can vary by patient, which makes research comparisons difficult. Baseline pain and functional limitations, BMAC dose, PRP dose (if given at the same time), and individual biologic factors can all influence treatment efficacy.

Some of the current indications and uses for BMAC under evaluation include chondral defects of the knee, full-thickness and moderate-to-large cartilage defects of the knee, osteoarthritis of the knee, meniscal tears, spine-mediated pain, partial tear of the rotator cuff tendon, ^, shoulder arthritis, osteochondral lesions of the talus, ACL repair, hip arthritis, and hip osteonecrosis. Autologous BMAC has also been used intraoperatively as an adjunct to debridement or microfracture surgery for cartilage defect and has been shown to improve healing and reduce rotator cuff retears when augmenting RTC surgery.

Studies of autologous BMC for the treatment of IA knee pathology are among the most numerous. Shapiro and colleagues performed a prospective, single-blind, placebo-controlled trial that compared BMAC and saline placebo injection for patients with bilateral knee osteoarthritis and found no serious adverse events from the BMAC procedures as well as a significant decrease in VAS pain scores at 1 week, 3 months, and 6 months.

With respect to the spine, there is an increasing number of studies. Pettine and colleagues examined outcomes in 26 patients following intradiscal percutaneous injection of BMC for discogenic low back pain. At 3-year follow-up, six patients went on to surgery. Of the remaining 20 patients, average ODI improved from baseline of 56.7 to 17.5 and average VAS improved from 82 to 22 at 36 months. At 1 year, 40% of patients had an improvement of one modified Pfirrmann grade on magnetic resonance imaging (MRI) and no patients had worsening of their imaging.

Caveats/Side Effects

Adverse reactions include general procedural risks, including but not limited to pain, swelling, bleeding, infection, damage to nearby structures, and potential detrimental effects of erythrocytes when used for IA joints. Contraindications include bone marrow–derived cancer (lymphoma), active systemic infection, blood thinners, and non-bone marrow–derived cancers which is a relative contraindication. However, a study by Hernigou and colleagues found no increased cancer risk in patients after application of autologous cell-based therapy using bone marrow–derived stromal progenitor cells either at the treatment site or elsewhere in the patients after an average follow-up period of 12.5 years.

Centeno and colleagues performed a large prospective study on 339 patients who were treated for various orthopedic conditions with culture-expanded autologous, bone marrow–derived MSCs that were harvested from the posterior superior iliac crest between 2006 and 2010. The mean patient age was 53 ± 13.85 years (214 males and 125 females) and follow up varied from 3 to 36 months. Using high-field MRI tracking and complications surveillance, they noted that while there were two patients who reported the development of cancer, the cancers were not at the injection sites and the percentage of patients that developed cancer was not far from the annual rate of cancer in the general population. The most common complaints were pain and swelling (2% of patients), with no reports of infections and procedure-related complications were self-limited or remediated with simple therapeutic measures. Centeno and colleagues subsequently performed a larger prospective multicenter study investigating the safety of same day autologous BMC aspiration isolation, and injection (n = 1590 patients, 1949 injections), same day autologous BMC aspiration, isolation and injection with an addition of minimally processed lipoaspirate (n = 247 patients, 364 injections), and cultured expanded BMSCs re-implanted weeks or months after bone marrow aspiration (n = 535 patients, 699 injections), orthopedics procedures across 18 sites. The discrepancy in the number of procedures and patients can be explained by multiple joint procedures that occurred during the same session, and/or serial injections occurring at different treatment sessions for the same patient. 3012 procedures were performed over 9 years with 2372 patients reporting on adverse advents (AEs) with a mean follow up time of 2.2 years. Follow-up ranged from 1 month to 8.8 years with an average of 2.2 years. ¹⁰⁷ A total of 325 (12.1%) AEs were reported, with 38 adjudicated to be definitely related to the procedure (1.6% of the population). The majority of the AEs were post-procedure pain or pain due to degenerative joint disease. Thirty-six (1.5% of the population) reported a serious side effect, with 19 of those adjudicated to be not related to the procedure and 4 definitely related. There were 7 neoplasms reported with none at the site of injections. The BMC only group was less likely to report AEs compared to the BMC with addition of minimally processed lipoaspirate and culture BMSC groups.

Blood-thinning medications such as coumadin and aspirin may be discontinued and managed appropriately by the cardiologist or primary doctor prior to the procedure. Lastly, the financial costs associated with BMAC procedures may be prohibitive for some patients as they are often not covered by insurance for elective orthopedic procedures.

FDA Regulations

There are two currently available BMSC preparations: cultured BMSCs and noncultured (nonexpanded) BMACs. The culturing method is currently prohibited in the United States as current regulatory guidelines consider in vitro expansion of the BMA more than “minimally manipulated.” In contrast, noncultured BMACs do not require laboratory cell expansion and culturing, and are available after centrifugation for same-day treatment. Given that this is a single-step process with “minimal manipulation” of cells, this meets the criteria established by the US FDA for homologous use. ^, As defined in 21 CFR 1271.3(c), “homologous use means the repair, reconstruction, replacement, or supplementation of a recipient’s cells or tissues with an HCT/P that performs the same basic function or functions in the recipient as in the donor.” Bone marrow meets this criterion for orthopedic uses as its constituents are involved in bone, cartilage, tendon, and muscle repair. Care should be taken when processing BMAC to not combine the BMAC with other agents, except for water, crystalloids, or a sterilizing, preserving, or storage agent to satisfy the minimally manipulated guidelines.

Minimally manipulated BMAC used for cartilage resurfacing therapies is not regulated by 21 CFR 1271 and therefore does not require premarket approval, preclinical research, or clinical trials to be marketed for treatment in the United States. ^, The FDA released a guidance document in November 2017 to explain the regulatory demands of manufacturers of human cells, tissue, and cellular and tissue-based products by specifically delineating the definition of “minimal manipulation.” ^, BMAC preparation kits and concentration systems require only a 510(k) Premarket Notification to the FDA 90 days before marketing the product. Thus, it highlights the importance of the clinician to counsel the patients effectively on the risks, benefits, and potential outcomes.

Mechanism of Action and Clinical Applications

MSCs are adult stem cells that can differentiate into a variety of cell types, including osteoblasts, chondrocytes, and adipocytes. ^, MSCs assist with tissue regeneration by differentiating into damaged cell types or indirectly by stimulating angiogenesis, limiting inflammation, and recruiting local tissue-specific progenitor cells. MSCs also secrete growth factors and cytokines, including but not limited to prostaglandins, TGF-B1, nitrous oxide, IL-4, IL-6, and IL-10, and IL-1 receptor antagonist. ^, HCSs can support the vascular system by differentiating into blood cells and stimulating osteogenesis with direct contact with MSCs. A study by Ceserani and colleagues on MFAT harvested using Lipogems Systems reported that the MFAT has the capacity to induce vascular stabilization and even retain antiinflammatory effects.

A study by Vezzani and colleagues noticed that the microanatomy of MFAT is similar to that of intact adipose tissue, with capillaries, microvessels, and pericytes wrapped around endothelial cells; however, they noted a higher frequency of pericytes in MFAT than in manual lipoaspirate and SVF. It is notable that recent literature has suggested that in vivo the main functionality of MSCs may not be multipotency. While pericytes have been widely believed to function as MSCs, others speculate that all MSCs could be pericytes due to the locations and cell marker signatures. Crisan and colleagues observed the expression of MSC markers on the surface of perivascular cells, suggesting blood vessel walls may harbor a reserve of progenitor cells. Guimarães-Camboa and colleagues’ lineage-tracing experiments suggest that the plasticity observed in vitro, or following transplantation in vivo, may be due to the artificial cell culture environment.

Microvascular fragments from adipose tissue exhibit a high angiogenic activity, representing a rich source of MSCs, and can rapidly develop into microvascular networks. When the resulting MFAT is processed, such as with Lipogems, pericytes can be retained within the intact stromal vascular niche as well as retaining the MSCs.

AT-MSCs isolated from younger donors are anticipated to be a more useful cell source for tissue engineering and regenerative medicine applications. There is theoretical potential for cell-based therapies for the elderly by banking adipose tissue at a younger age, preserving the stem and progenitor cells when biologic activity is at its greatest potential.

Acknowledging a need for further high-quality research, the American Academy of Orthopaedic Surgeons in 2018 collaborated and called for establishing high-quality patient registries that can assist with continued surveillance and quality assessment.

Caveats/Side Effects

Adverse reactions include general procedural risks including but not limited to pain, swelling, bleeding, infection, and damage to nearby structures. Kuah and colleagues evaluated the safety and tolerability of in vitro expanded MSCs derived from human donor adipose tissue combined with cell culture supernatant, and did not find any severe side effects when administered as a single IA injection to patients with symptomatic knee osteoarthritis. Swelling of injected joints can be a common side effect, and is thought to be associated with the death of stem cells. Other potential side effects, though less common, include tenosynovitis and tendonitis, arthralgia, joint stiffness, effusion, and paresthesia or malaise. ^{, ,}

Theoretically, MSCs can divide into unwanted oncologic cell lineages, and could contribute to tumor behavior by influencing the tumor microenvironment and promoting angiogenesis; however, this has not been seen with adult-derived MSCs. No literature documenting neoplastic complications at ADSC implantation sites have been reported, and a longitudinal cohort study by Pak and colleagues on SVF with PRP into various joints found no evidence of neoplastic complications in any implantation site.

The cost of the procedure is generally not covered by insurance, and can be prohibitive for some.

FDA Regulations

There are some FDA-compliant devices that can make MFAT with intact stromal vascular niche and MSCs such as Lipogems, PureGraft, and IntelliFat, which can then be injected back into the patients through the use in orthopedic applications, and whether it counts as homologous use is debatable. The FDA defines the basic function of adipose tissue as providing cushioning and support. Some argue MFAT acts to cushion and/or support orthopedic tissue injuries, such as tendon and meniscal tears, and should be considered homologous.

Recently published guidelines that indicate enzymatically or mechanically processed lipoaspirate (PLA) alters these relevant characteristics and is not considered homologous use or minimal manipulation by the FDA. ^, SVF is adipose tissue that undergoes further processing (such as with enzymatic digestion) to break down and eliminate the adipocytes and surrounding structural components that provide cushioning and support, and thus is considered more than minimally manipulated. ^, Further laboratory cell sorting and culture expansion would also be considered more than minimally manipulated due to the steps involved in isolating and culturing of cells. Adipose tissue that is processed into SVF or cultured ASC is more than minimally manipulated and not FDA approved based on the current guidelines. Minimally manipulated ADSC used for cartilage resurfacing therapies is not regulated by 21 CFR 1271 and therefore does not require premarket approval, preclinical research, or clinical trials to be marketed for treatment in the United States. Consequently, some products are not FDA approved and may qualify instead for investigational new drug process.

Bone Marrow–Derived Versus Adipose-Derived Stem Cells

MSCs can be obtained from different sources, including bone marrow, adipose tissue, dental pulp, synovium, muscle, and other tissues. To date, bone marrow–derived MSCs are the best characterized and studied. Recently, other MSC sources, particularly adipose tissue, have gained clinical interest for use in regenerative medicine. ^, Zuk and colleagues were one of the initial researchers to assess PLA cells obtained from liposuctioned adipose tissue, and found it represented a potential source of multilineage mesodermal stem cells. Few studies have compared the efficacy of BMAC and MFAT. ^,

There are vast differences in the reported stem cell yields in adipose and bone marrow, with some studies reporting 100 to 500 times higher stem cell numbers found in adipose versus bone marrow. However, based on donor-matched studies the differences volume per volume or gram for gram appear far less than that. In one study, patients undergoing ACL reconstruction donated samples of several tissues including bone marrow and adipose. Bone marrow had about 93× more TNCs per volume compared to adipose (2045 vs 22). Adipose had about 148.5× more CFUs per 1000 TNCs, but bone marrow had the most cells per colony. Given that bone marrow had more TNCs, adipose contained about 1.6× as many MSCs per volume compared to bone marrow. BMC was also found to contain 6x more nucleated cells compared to SVF. SVF contained 4× more adherent cells. Colony-forming frequency was 0.5% in SVF versus 0.01% in BMC. MSC population was 4.28% of the TNC in SVF versus 0.42% in BMC or about 10× more in SVF. In summary, bone marrow has more nucleated cells than adipose but only a small percentage are MSCs. Adipose has less nucleated cells but a higher percentage of those are MSCs. Pending variability in harvesting techniques and patient characteristics, adipose per volume likely has 1.6 to 10× as many MSCs as bone marrow. It has been reported that the lipoaspirate harvesting, when compared to bone marrow harvesting, can yield more absolute MSCs, the MSCs have faster rapid in vitro expansion, and may yield more stem cells per gram of tissue. ^{, ,}

While there are similar and varying cell surface marker profiles between bone marrow–derived mesenchymal stem cells (BM-MSCs) and adipose tissue–derived mesenchymal stem cells (AT-MSCs), they both have the potential to differentiate into multiple lineages, including osteogenic, chondrogenic, adipogenic, cardiomyocytic, hepatic, and neurogenic differentiation. ^{, ,}

BM-MSCs and AT-MSCs have comparable immunophenotypes; however, AT-MSCs retain their phenotype more consistently over BM-MSCs across multiple culture passages, and also have higher proliferation capacities in part due to their lower senescence ratio. A donor-matched study comparing culture-expanded AT-MSCs vs BT-MSCs showed that AT-MSCs underwent faster proliferation and doubling times. They had longer life spans possibly due to the fact they had longer telomeres, which may suggest a smaller dose is required in vivo. Both had similar differentiation capacity and decreased proliferation the longer the in vitro expansion was performed. On the other hand, Mohamed-Ahmed and colleagues found BM-MSCs were superior to AT-MSCs in terms of osteogenic and chondrogenic differentiation, while AT-MSCs had higher proliferation and adipogenic potential. Chondrogenesis was more efficient in human BM-MSCs versus AT-MSCs in vitro. Human BM-MSCs showed more chondrogenic potential in vitro compared to AT-MSCs. Xu and colleagues noted that BM-MSCs possessed stronger osteogenic and lower adipogenic differentiation potentials compared to AT-MSCs, but found no significant difference in the chondrogenic differentiation potential. Xu and colleagues’ results demonstrated that the DNA methylation status of the main transcription factors controlling MSC fate influenced their expression, and subsequently different differentiation capacities of AT-MSCs and BM-MSCs.

Clinically, there are limited clinical data comparing BMAC to MFAT. Mautner and colleagues conducted a retrospective study of 110 patients comparing the pain and functional outcomes of patients with symptomatic knee osteoarthritis who received BMAC or MFAT or injections. At a mean follow-up time for BMAC of 1.8 years and 1.0 years for MFAT, they noted both groups had significant improvement in Emory Quality of Life, VAS for pain, and Knee Injury and Osteoarthritis Outcome Score questionnaire ( P < .001) without a significant difference between BMAC and MFAT.

Mesenchymal Stem Cells/Stromal Cells From Bone Marrow

After harvesting BMA, the sample is processed in a centrifuge and subsequent buffy coat layer and platelet-poor plasma layers are removed. The cells are subsequently isolated, generally by their adherence to plastic, and then cultured. ^, Culturing methods can vary, though one method by DiGirolamo and colleagues includes diluting the aspirate marrow with Hank’s balanced salt (HBS; Gibco), washing gently with inversion, adding a layer of Ficoll (Ficoll‐Paque; Pharmacia) beneath the sample, and then spinning the sample at 2500 g for 30 minutes at room temperature. The mononuclear cell layer was then washed again with HBS and the cells spun at 1500 g for 15 minutes and resuspended in complete medium (Minimum Essential Medium, alpha medium without deoxyribonucleotides or ribonucleotides, Gibco; 20% fetal calf serum lot‐selected for rapid growth of human marrow stromal cells (hMSCs) FCS, Atlanta Biologicals; 100 units/mL penicillin, 100 μg/mL streptomycin, Gibco; and 2 mm L‐glutamine, Gibco). Cells were then plated in a 25 cm ² tissue cultured flask (Nunc), incubated at 37°C with 5% humidified CO ₂ for 1 day before the non-adherent cells were removed. Adherent cells were then washed twice with PBS and shaken to remove adherent hematopoietic precursors, and fresh complete medium was added. The medium was replaced every 3 to 4 days until the cells grew to 70% to 90% confluency, and harvested with trypsin and ethylenediaminetetraacetic acid (EDTA), with the entire process generally repeated to expand the cells through various passages.

Mesenchymal Stem Cells/Stromal Cells From Adipose Tissue

After harvesting adipose tissue, the raw lipoaspirate can be processed using various methods and protocols to produce an injectable, including MFAT, enzyme-digested SVF, or cultured ASC. ^, After isolation from the raw lipoaspirate (refer to formulation section in Adipose Tissue section above), the SVF can either be used directly in clinical procedures or can be cultured to increase the number of cells before using them clinically. With cell culturing, only ASCs and their precursor cells (supra-adventitial cells and pericytes) are able to adhere and survive. ^, It is thought that the predominant benefit of SVF relies on the presence of ASCs. The process of isolating and culturing ASCs is generally a long procedure that is not performed at the same time as surgery. ^,

When the adipose tissues are further processed into the SVF or cultured ASC, they are considered more than minimally manipulated and do not meet the criteria established by the FDA and do not comply with current regulations. To make SVF, the raw lipoaspirate is washed with PBS and subsequently undergoes enzymatic digestion with collagenase, dispase, trypsin, or other enzymes (ranging from 30 minutes to 1 to 2 hours). After neutralization of the enzymes, the remaining elements are now called the stromal vascular fraction or SVF, and are separated from the mature adipocytes by centrifugation. The SVF is composed of a heterogeneous mixture of cells, including pre-adipocytes, pericytes, mast cells, fibroblasts, smooth muscle cells, endothelial cells, hematopoietic stem cells, and AT-MSCs. ^,

When SVF cells are cultured, a subset of cells adheres to the tissue culture plasticware. After purification with washing and culture expansion with media, the HCSs (nonplastic adhesive cells) are separated from the SVF cells, leaving behind the adipose tissue–derived stromal cells which include the stem cells, which can be expanded in a culture medium. ^, The cells are then resuspended in a mixture of culture medium and cryoprotective medium and frozen and stored under good manufacturing practice. Cells can then be expanded in a culture medium for extended periods and utilized later. ^, Digesting the triple helix region of the peptide bonds in the collagen of adipose tissue with collagenase can be both time consuming and expensive. In some cases, it may be possible to obtain ASCs without enzymatic digestion. ^, Baptista and colleagues isolated a population of MSC from lipoaspirate samples without tissue digestion and found that it is possible to store freshly isolated mechanically processed lipoaspirate adipose tissue (MPLA) cells by cryopreservation without a significant loss of MSCs.

Jurgens and colleagues investigated whether the yield and functional characteristics of ASCs are affected by the adipose tissue-harvesting site and reported that the percentage of ASCs in the SVF of adipose tissue harvested from the abdomen was significantly greater than those harvested from the hip/thigh region ( P < .05). The authors noted that while there not was a significant difference in the ASC proliferation or differentiation capacity from cells harvested in the abdomen compared to cells harvested from the hip and thigh, there was a significantly increased yield of ASCs found in SVF of abdominal adipose tissue compared to SVF of adipose from hip/thigh regions. Furthermore, Iyyanki and colleagues similarly found higher total SVF, but not tissue–derived stem cell yields in fat harvested from the abdomen compared to the flank or axilla. Tsekouras and colleagues assessed 53 lipoaspirates in various locations (inner thigh, outer thigh, abdomen, waist, and inner knee) and found that the outer thigh exhibited significantly higher SVF cell count compared to other donor sites and both the inner and outer thigh showed a significantly higher number of ASCs. No significant differences in the viability of SVF cells and ASCs were noted. Fraser and colleagues evaluated the stem and progenitor cell content of subcutaneous adipose tissue in the hips and the abdomen of 10 subjects undergoing elective liposuction. Using clonogenic culture assays and a paired analysis of tissue obtained from the different subcutaneous sites, the authors noted that tissue harvested from the hip yielded a statistically significant 2.3-fold more fibroblast CFU volume and a 7-fold higher frequency of alkaline phosphatase-positive colony-forming unit (CFU-AP, a widely used marker for osteogenesis) than that obtained from the abdomen. One caveat is that the results could reflect the differences in ratio of deep and superficial subcutaneous adipose tissue collected from the two sites, as deep subcutaneous adipose tissue generally has a higher frequency of blood vessels, and thus more pericytes which may be detected by the CFU-AP assay Pericytes, as mentioned before, have been shown to possess an adipogenic potential.

FDA Regulations

Cultured MSCs are currently prohibited in the United States, as the methods for culture and isolation are currently considered more than minimal manipulation of the cells. The culturing method is performed outside of the United States; however, some institutions through special approval for research have been able to utilize culture-expanded cells. The FDA released a guidance document in November 2017 to explain the regulatory demands of manufacturers of human cells, tissue, and cellular and tissue-based products by specifically delineating the definition of “minimal manipulation.”

Conclusion

Culture-expanded MSCs certainly have potential in clinical applications; however, more robust science is necessary to better understand their safety profile and efficacy in musculoskeletal applications.