Introduction

Regenerative medicine is an emerging field that harnesses the body’s own healing capacity to enhance tissue recovery, decrease pain, and improve functionality. Since the first use of the human body’s own cells for hematopoietic stem cell transplantation in 1957,1 several historical advancements have been achieved in the field of regenerative medicine including: allogeneic skin tissues in 1998,2 gene therapy to treat severe combined immunodeficiency (SCID) in 1990,3 and development of cellularized scaffolds to replace tissues in 2016.4 Similarly, the integration of regenerative medicine into the field of pain has also surged over the past decade, supported by preclinical and clinical studies.

With the goal of treating painful conditions, non-cellular regenerative medicine modalities have also been developed, including prolotherapy in the 1930s, followed by platelet-rich plasma (PRP) in the 1990s for orofacial wound management,5,6 both of which have rapidly gained traction, mainly in sports medicine and orthopedic surgery. Furthermore, the identification of mesenchymal stem cells (MSCs) has introduced another promising biologic and novel avenue for clinical use.

Regenerative medicine comprises approaches such as cell-based therapy, gene therapy, and tissue engineering that influence cell proliferation, interaction, and extracellular matrix restoration. Within pain management, regenerative medicine is employed as a treatment for a spectrum of conditions ranging from musculoskeletal conditions to neuropathic pain to spinal cord injury. Proposed mechanisms of action involve mobilizing inflammatory cells and secretion of cytokines and growth factors that play pivotal roles in tissue regeneration and maturation, and acting as natural anti-inflammatory agents.7 The pillars of regenerative medicine treatments in pain management include non-culture-expanded autologous MSCs from products such as bone marrow aspirate concentrate (BMAC) or fat aspirate, PRP, and their derivates (Figure 1). Per federal guidelines, these products should meet the requirement of minimally manipulated products. Currently, only 36 more than “minimally manipulated” cell and gene therapies are approved by the Food and Drug Administration (FDA), primarily targeting cancer, inherited genetic diseases, and allogeneic umbilical cord cell transplantation.8

Figure 1 Diagram displaying adult and perinatal sources of mesenchymal stem cells. Adapted with permission from Hoang DM et al. Stem cell-based therapy for human diseases. Signal Transduct Target Ther. 2022 Aug 6;7(1):272.9

Utilization of regenerative medicine therapy for pain in the absence of widely accepted consensus guidelines leads to inconsistency in practice, reliance on physician experience, case-by-case decision-making, and variable interpretation from outcomes of small, heterogeneous trials with mixed results. This inconsistency impedes the broad acceptance of regenerative medicine by physicians, government regulatory agencies, and health insurance providers, potentially hindering patient access to beneficial treatment.

Therefore, the American Society of Pain and Neuroscience (ASPN) commissioned a group of experts to create formal guidelines summarizing the existing evidence for regenerative medicine treatment for chronic pain indications. Notably, the American Society of Interventional Pain Physicians (ASIPP) previously issued guidelines on biologics for low back pain10 and the American Academy of Orthopedic Surgeons (AAOS) issued an overview on the use of PRP for knee osteoarthritis (OA),11 although neither addressed the full spectrum of regenerative medicine therapy in chronic pain management. Our objective was to develop pragmatic guidelines that can guide clinical care and establish a basis for future clinical research and regulatory agencies’ considerations.

Materials and Methods Development Process

We utilized a systematic approach to appraise the evidence on regenerative medicine interventions (eg, injectable biologics) for treatment of chronic pain, including the type of injectable biologics, evidence for various chronic pain indications, best peri-procedural practices to optimize response to injectable biologics as well as yield and quality of injectable biologics, safety profile and adverse effects, and ethical considerations. We employed a similar methodology that was utilized in prior guideline articles produced by ASPN.12–14 This current consensus guideline for regenerative medicine interventions adds to the current family of consensus practice guidelines and incorporates a systematic review process.

To create this consensus guideline, the ASPN society commissioned an international multidisciplinary panel consisting of anesthesiologists, pain medicine physicians, physical medicine and rehabilitation physicians, and sports medicine physicians who are experts in the field of regenerative medicine. To qualify for inclusion on this multidisciplinary panel, each author was required to have experience with offering and performing regenerative medicine therapy, or publications or ongoing research pertaining to regenerative medicine. Further, each panel member’s impact on the field of regenerative medicine was considered.

A steering committee was tasked with selecting preliminary questions, which were reviewed and refined to 16 questions by the entire committee. Questions were assigned to 3 to 4 person modules, who collectively worked with the Committee Chairs (RSD, TRD) on preliminary versions. After completion of all sections for each question, this was subsequently distributed to the full committee.

Regenerative medicine continues to be a relatively novel and emerging field, with evolving evidence. While systematic review methodology15–18 is standard for forming consensus guidelines on established therapy, the development of new modalities, disease applications, and other innovations may also benefit from this approach and advance the field faster than randomized controlled trials (RCTs). Therefore, in instances where RCT-level data were unavailable, the authors reviewed the available evidence with lower levels of quality coupled with clinical expertise from the multidisciplinary panel to fill the gap. In this section, we outline the methodology including the search strategy, systematic review process, and consensus guideline creation and grading.

Literature Search Methods

A protocol was developed a priori outlining steps to be undertaken in this consensus guideline. A comprehensive search of several databases was performed on November 14, 2023, identifying relevant studies on regenerative medicine therapy for chronic pain. No date or language limits for the search were applied. Databases searched (and the content coverage dates) were Ovid MEDLINE(R) (1946+ including epub ahead of print, in-process, and other nonindexed citations), Ovid Embase (1974+), Ovid Cochrane Central Register of Controlled Trials (1991+), and Scopus via Elsevier (1970+). The search strategies were designed and conducted by a medical librarian (LH) with input from the study investigators. Controlled vocabulary supplemented with keywords was used. The actual strategies listing all search terms used and how they were combined are available in the Supplemental Material. We also hand-searched reference lists of identified publications to identify relevant publications not captured in the librarian-assisted search strategy.

Broad MeSH terms and Boolean operators were selected for each database search, including terms and synonyms for regenerative medicine, stem cells, injectable biologics, mesenchymal stem cells, platelet-rich plasma, bone marrow aspirate concentrate, autologous conditioned serum, stromal vascular fraction, adipose-tissue derived biologics, analgesia, chronic pain, neuropathy, and other specific chronic pain conditions.

Inclusion criteria encompassed studies that described regenerative medicine (eg, injectable biologics) for treatment of various pain conditions (eg tendinopathy, OA, muscle pathology, conditions manifesting with neuropathic pain, and other selected conditions), physiological mechanism of action of injectable biologics, technique of obtaining injectable biologics, adverse events, ethical considerations, and other aspects. Study designs that were identified and included in our search strategy included RCTs, prospective observational studies, and retrospective observational studies. If RCTs or observational studies were unavailable for certain questions addressed in this guideline, we utilized case series, case reports, or pre-clinical studies. Given the emerging body of the literature pertaining to regenerative medicine, the panel felt that these sources of evidence also held value to help inform consensus guidelines. Exclusion criteria comprised non-peer reviewed publications and certain study designs (eg, review articles). Two authors (YFH and MER) independently selected abstracts as well as full-text articles from the above listed databases using the aforementioned search strategies. A third author (RSD) adjudicated any discrepancies.

Systematic Evaluation of Evidence and Consensus Best Practices Development

A total of 8256 studies were identified with the initial search. Abstracts for each study were reviewed independently by two authors (YFH and MER) to identify studies for full-text review. In total, 970 articles were identified for full-text review and, of these, 740 articles were included for final analysis.

Given the infancy of regenerative medicine and its emerging literature, the primary goal was to fill in gaps of knowledge through consensus from our panel. Based on the reviewed evidence, a list of consensus points was created by the panel. Evidence for each consensus point was appraised using the United States Preventive Services Task Force (USPSTF) criteria for evidence level (Table 1) and degree of recommendation (Table 2). A degree of recommendation indicates the highest degree of recommendation, while D indicates the lowest degree of recommendation. A list of all consensus points reported in this guideline is included in Table 3.

Table 1 Hierarchy of Studies by the Type of Design (US Preventive Services Task Force)

Table 2 Meaning of Recommendation Degrees (I.S. Preventive Services Task Force)

Table 3 List of Consensus Points

Modified Delphi Approach and Oversight of Bias and Conflict of Interest

All authors disclosed financial conflicts of interest and were asked to recuse themselves on any issue with which they have a relationship as well as competing interests. Conflicts of interest were disclosed and managed before initiation of the guideline development process. Per ASPN protocol,20 one of the primary authors was non-conflicted (RSD) and served as the editor of the paper for bias.

Using a modified Delphi approach, the interim draft and consensus points were distributed to all collaborators en bloc and each collaborator was requested to vote on each point.21 The evaluation criteria were simplified with three potential options: “agree” or “disagree” or “abstain”. If collaborators provided a decision of “disagree” or “abstain” for any point, they were invited to provide a rationale for their decision in a non-blinded fashion to the committee chair (RSD), who incorporated the respective comments and distributed revised versions to the full committee until consensus was achieved.

Two rounds of voting were implemented. Of those allowed to vote on consensus points, an eighty percent agreement rate was required to accept the consensus point. However, recommendations with <100% agreement were revised based on feedback from those who disagreed or abstained from voting. During the second round of voting, consensus points with a pre-defined >80% agreement were accepted. In addition, the list of final consensus points and manuscript were shared with all collaborators for final approval.

Question 1: What Injectable Biologic Agents are Available for Treatment of Chronic Pain Conditions?

In this section, we provide details on specific injectable biologics including PRP, autologous-conditioned serum (ACS), bone marrow aspirate concentrate (BMAC), adipose tissue-derived biologics, mesenchymal stem cells, amniotic-based products, hollow osseous fillers, extracellular matrix substances, and extracellular vesicles. Details on preparation and processing of all injectable biologics are provided in Supplemental Table 1. A list of 501(k) cleared preparation systems and FDA approval status are provided in Supplemental Table 2. In addition, a list of companies that offer regenerative medicine therapy are displayed in Supplemental Table 3.

Platelet Rich Plasma

PRP is a blood product derived from the centrifugation of a patient’s whole blood to produce a supraphysiologic concentration of platelets suspended in blood plasma. The PRP product is then injected into the site of injury with the goal of supplementing the body’s natural healing mechanisms. Initial uses for PRP were found in dentistry and oral maxillofacial surgery as well as wound healing in dermatology, although its uses have substantially expanded to musculoskeletal disorders.22 The mechanism of PRP involves potentiation of the healing process that is mediated by the release of growth factors including epidermal growth factor (EDF), insulin-like growth factor one (IGF-1), basic fibroblast growth factor one (bFGF-1), platelet-derived growth factor alpha/beta (PDGF-alpha/beta), vascular endothelial growth factor (VEGF), and transforming growth factor beta1 (TGF-β1), amongst others. Additionally, the release of other bioactive molecules such as metabolites, chemokines, cytokines, and dense granules of platelets supplement the growth factor response.23,24 Of all injectable biologics, PRP is most commonly studied and evidence generally supports its use to treat pain from OA and ligament injuries. A list of injectable biologics, their key characteristics, and respective secreted biochemical factors are displayed in Supplemental Table 4.

Autologous Conditioned Serum (ACS)

ACS is a product that was first introduced in the 1990s and used primarily to treat musculoskeletal injuries. It is derived from incubating venous blood under controlled conditions (historically within specialized glass spheres), which promotes monocyte activation and leads to high levels of several anti-inflammatory cytokines in the product including interleukin (IL) 1Ra, IL-10, and TGF-β1.25,26 There is recent evidence that the analgesic and functional improvements with ACS are due to exosome expansion during the incubation process.27 Studies have highlighted the efficacy of ACS in treatment of musculoskeletal disorders, with OA being the most frequent indication, followed by soft tissue pathology such as tendinopathies and radiculopathies.25,28–31 The preparation of ACS involves more than minimal manipulation and therefore this product is not approved for use in the United States.

Bone Marrow Aspirate Concentrate (BMAC)

There are two types of bone marrow: red and yellow. Red bone marrow contains hematopoietic stem cells (HSCs) that give rise to red blood cells, white blood cells, and platelets. Yellow bone marrow is made mostly of adipocytes and contains stem cells that can become cartilage, fat, or bone cells. The bone marrow can produce 200 billion red blood cells, 10 billion white blood cells, and 400 billion platelets on a daily basis.32 BMAC contains a variety of progenitor cells, including HSCs, endothelial progenitor cells (EPCs) and mesenchymal stromal cells (MSCs, also known as “mesenchymal stem cells” or “medicinal signaling cells”). The frequency of these progenitor cells in whole blood is very low, unless a trauma has occurred. The use of bone marrow-derived cells in the treatment of orthopedic conditions has been shown to be secondary to both MSC and HSC effects. Furthermore, MSCs are believed to be pericytes, or, perivascular cells, with different populations and properties based on different vascular niches. These cells localize adjacent to marrow vessels and can be identified by different markers besides the typical mesenchymal markers (cluster of differentiation [CD]105, CD90, CD73).33 The process of extracting and processing BMAC is described in Supplemental Table 1 and is displayed in Figures 2–4.

Figure 2 Diagram displaying bone marrow aspiration technique. Two approaches to bone marrow aspiration are displayed in this figure. We advise the approach from the posterior superior iliac spine to the anterior inferior iliac spine (novel approach) due to its safety margin away from the greater sciatic notch. Adapted with permission from D’Souza et al. A three-dimensional computed tomography study to determine the ideal method for fluoroscopically guided bone marrow aspiration from the iliac crest. Bosn J Basic Med Sci. 2021 Jun 1;21(3):370–377.34

Figure 3 Diagram displaying bone marrow aspiration. Fluoroscopic imaging of the posterior iliac crest with the Jamshidi needle. A hammer is used to gently penetrate the cortex of the iliac crest prior to aspiration.

Figure 4 Processing of bone marrow aspirate to create bone marrow aspirate concentrate. Note the three fractions of the bone marrow aspirate after centrifugation. The fraction of interest is in the middle buffy coat directly above the dark red blood cells.

Adipose Tissue-Derived Biologics

Adipose tissue is a readily available source of adipose-derived cells (ADCs) which contain a heterogeneous population of cells including MSCs, EPCs, regulatory T cells, macrophages, smooth muscle cells, pericytes and preadipocytes. Extraction of adipose tissue is performed by liposuction. Approximately 400,000 liposuction surgeries are performed in the United States each year. Liposuction can yield between 100 mL to >3 L of lipoaspirate tissue, which is routinely discarded.35 In general, two categories of adipose tissue are derived to concentrate MSCs: stromal vascular fraction (SVF), micro-fragmented adipose tissue (MFAT), and more recently nanofat.

SVF is a heterogeneous collection of cells contained within adipose tissue that requires the use of enzymes such as collagenase, dispase, or trypsin to break down the connective tissue. With the removal of connective tissue, there is a greater concentration of SVF in the aspirate made up of MSCs, EPCs, regulatory T cells, macrophages, smooth muscle cells, pericytes and preadipocytes. The following terminology has been used to identify the same adipose tissue cell population that is derived after enzymatic processing: Adipose-derived Stem/Stromal Cells (ASCs); Adipose Derived Adult Stem (ADAS) Cells, Adipose Derived Adult Stromal Cells, Adipose Derived Stromal Cells (ADSC), Adipose Stromal Cells (ASC), Adipose Mesenchymal Stem Cells (AdMSC), Lipoblast, Pericyte, Pre-Adipocyte, Processed Lipoaspirate (PLA) cells. The use of this diverse nomenclature has led to significant confusion in the literature.36

MFAT is a mechanical fragmentation of adipose tissue that avoids the use of enzymes. Utilizing metal beads or progressively smaller sieves, the lipoaspirate is refined to clusters of 0.2–0.8 mm. Repeated washing with saline is performed to remove lipids, blood, and tumescent solution. Techniques that have compared purely mechanical breakdown of adipose tissue, compared to the addition of collagenase, have demonstrated up to a 5-fold increase in MSCs favoring the addition of collagenase.37–41

Nanofat is an emerging ADC that is derived from emulsification and filtration of lipoaspirate.42,43 Components of nanofat approximate 400 to 600 μm. In nanofat, the product is devoid of mature adipocytes and contains microvascular fragments of arterioles, venules, and capillaries in addition to a variety of growth factors, biological peptides, and cytokines.

A disadvantage of ADCs is the processing of SVF, which is currently not allowed by the FDA. Processing of adipose tissue to create SVF is considered more than a minimally manipulated product, and thus constitutes a drug.44 Risks of liposuction include soft tissue and vascular damage, therefore requiring further training not common to the skillset of an interventional pain physician or sports medicine physician. Further, there may be a need for a liposuction system, although the quantity of lipoaspirate needed for adipose processing can be easily performed with a vacuum-locked syringe system.

Mesenchymal Stem Cells

Arnold Caplan first coined the term MSCs for their ability to differentiate into cells of the mesodermal lineage.45 The International Society for Cellular Therapy proposed a minimum criteria requirement for cells to be classified as human MSC. The cells must be able to differentiate in vitro into chondrogenic, osteogenic, or adipogenic tissue. They must express CD105, CD73, and CD90 but not express Human Leukocyte Antigen (HLA)-DR molecules, CD14, CD34, CD11b, CD45, CD79a, or CD19. Lastly, these cells must be able to adhere to plastic when preserved in standard culture conditions.46 MSCs are further classified as bone marrow-derived MSCs (BM-MSCs), adipose-derived MSCs (A-MSC), although other sources are reported as well.47 BM-MSCs are typically aspirated and harvested from bone marrow in the iliac crest or hip while A-MSCs are harvested from subcutaneous adipose tissue, often from the abdomen. Umbilical and amniotic fluid-derived MSCs have been proposed but are not as well-studied.48 Adult and perinatal sources of MSCs are displayed in Figure 1.

The therapeutic activity of MSCs is mediated by paracrine effects. It has been proposed that these cells secrete a variety of anti-inflammatory cytokines.49,50 Whereas the common misconception is that the stem cells will identify injured tissues and differentiate directly into those tissues by engraftment, it has been demonstrated that less than 1% of stem cells injected remain after as little as 7 days.51,52 MSCs respond to injured tissues and can coordinate a healing response via secretion of bioactive molecules, such as angiopoietin (Ang)-1, Ang-2, bone morphogenetic protein (BMP), brain-derived neurotrophic factor (BDNF), IL-6, and VEGF.53 MSCs have immune-privileged status, multilineage potential and the ability to support angiogenesis in a paracrine fashion.

Amniotic-Based and Other Perinatal Products

Initially utilized for treatment of burns and wound healing, amniotic products including amniotic membranes and fluid have been suggested for use as injectable biologics.54 Amniotic membrane consists of amniotic epithelial cells and amniotic MSCs.55 The latter consists of fibroblasts as well as protein complexes that collectively facilitate cellular transport. Amniotic membranes are thought to regulate amniotic fluid, form a protective layer for the fetus, and secrete bioactive molecules.55 These include fibroblast growth factors, epidermal growth factor, hyaluronic acid (HA), IL-1, IL-10, beta-defensins, TGF-β1, elafin, HLA-G, matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases, and PDGF.55,56 Their anti-inflammatory and metabolically active properties allow for catabolism and subsequent tissue regeneration. Other perinatal products in use include umbilical cord blood and Wharton’s jelly.

Extracellular Matrix (ECM) Substances

Similar to biologic osseous fillers, extracellular matrix, also known as demineralized bone matrix (DBM), is allograft bone tissue with osteoconductive and osteoinductive properties. Harvested from donors, DBM contains mostly type 1 collagen, some type IV and type X collagen, as well as non-collagenous proteins and various osteoinductive growth factors (BMP, insulin-like growth factors, and PDGF).57 Injected or placed along bone sites of injury requiring growth and repair, the DBM is broken down by the host cells resulting in a release of growth factors promoting bone growth.58 Once obtained, DBM can be further augmented to provide a combinatorial effect, such as therapeutic drugs or bioactive materials, to enhance or augment the intrinsic activity of DBM.58

Recombinant Bone Morphologic Proteins

Bone morphogenetic protein-2 (BMP-2) is one of two recombinant BMPs utilized for bone induction in patients. Recombinant BMPs are used because endogenous human BMP from cadaveric bones have insufficiently small yields needed for clinical applications.59 BMPs are a group of proteins in the TGF-β family that bind to cell surface serine-threonine kinase receptors activating gene expression for bone creation.60 Cell culture cell lines are utilized to create recombinant BMP-2. Though effective, BMP-2 use has been controversial as it has been associated with possible neurologic impairment, heterotrophic ossification, wound complications, and carcinogenicity.61,62

Extracellular Vesicles

Extracellular vesicles (EVs) are promising therapeutic agents that are derived from platelets and progenitor cells and have been reported to support the paracrine function associated with progenitor cells.63 EVs are small particles (50 nm to 1000 nm) released by cells in the body, are involved in cell-to-cell communication in vivo and are found in all fluids and tissues in the body.64 While the mechanisms of potential therapeutic benefit of EVs in vivo are under evaluation, there is developing literature for MSC exosomes and platelet exosomes in the treatment and restoration of cartilage and even nerve injury.65,66 MSC-exosomes, the smallest class of EVs and derived from MSCs, when added to cultured nucleus pulposus cells obtained from a degenerated disc resulted in reestablishing a normal nucleus pulposus cell phenotype of the degenerated disc, suggesting its regenerative potential.67

Therapeutic Mechanisms of Injectable Biologics

There are several important sources of potential therapeutic benefit associated with injectable biologics. This section focuses on the common mechanisms of PRP, BMAC, and ADCs (eg, SVF, MFAT):

All three preparations release EVs. The released EVs bind to target tissue-resident cells and are known to mimic the paracrine function of progenitor cells and platelets.63 EVs also amplify the therapeutic benefit of the cells and platelets present in injectable biologics. PRP and BMAC contain plasma, which is known to contain beneficial cytokines and functional biomolecules like IL-1Ra and α-2-Macroglobulin (a2M). IL-1Ra blocks the binding of IL-1β, which reduces pain associated with the pro-inflammatory supporting IL-1β molecule.68 a2M is an inhibitor of proteolytic enzymes like trypsin, but more importantly inhibits the collagen-degrading enzymes known as MMPs, including Collagen Type II-degrading MMP-13.69 Platelets release growth factors and cytokines (the paracrine effect) following activation, including Epidermal Growth Factor (EGF), VEGF, Insulin-like Growth Factor (IGF), TGF-β1 and TGF-β2, adenosine, serotonin, and PDGF-BB, among others.70,71 These factors are able to promote proliferation of resident cells, enhance stem/stromal cell homing to the site of pathology, and reduce inflammation in the injected site.72 Progenitor cells are able to respond to their environment and release a wide variety of bioactive molecules, including molecules that have important activities for enhanced tissue cell proliferation, immunomodulation, neo-angiogenesis, wound repair, rescue of dying cells (anti-apoptotic) and anti-microbial activity.73,74 Mononuclear cells obtained from whole blood (eg, peripheral blood-derived mononuclear cells) that contain monocytes might provide a therapeutic benefit, since monocytes are known to transdifferentiate into anti-inflammatory macrophages (Type 2, M2).75 Monocytes are enriched in PRP and BMAC.

Although both the platelets in PRP and the progenitor cells in BMAC and SVF participate in the paracrine mechanism and release EVs and cytokines, once the platelets in PRP degranulate, there is no further therapeutic benefit. In contrast, progenitor cells are able to respond to their microenvironment74 and continue to release growth factors, cytokines and EVs after being delivered to the pathologic site.63 Thus, the therapeutic benefit when treating with cell-containing injectable biologics might theoretically be more robust and dynamic compared to that achieved by platelets in PRP. The therapeutic components present in injectable biologics primarily are focused on reducing the pro-inflammatory milieu within the injected site, to rebalance the catabolic and anabolic pathways. Restoring homeostatic balance may allow for the resident cells to return to their pre-degenerative status, improve the flux of nutrients, and stabilize the mechanical loading properties.76

Consensus Point 1. Patients should be advised that the mechanisms of action of injectable biologics in the treatment of chronic pain conditions are multifaceted and related to the specific injected biologic agent. Most mechanisms are centered on modulation of the injected tissue to promote an anti-inflammatory microenvironment. Common mechanisms include (1) release of anti-inflammatory cytokines, (2) release of growth factors, (3) differentiation of mononuclear cells into anti-inflammatory macrophages, and (4) release of extracellular vesicles that bind to target tissue resident cells and perform a paracrine function similar to progenitor cells (Level II-2, Grade C).

Question 2: What is the Evidence Basis for Variants of Standard Platelet Biologics?

Due to the variability of the different PRP products and production protocols (eg, preparation, dosing, delivery), as well as lack of clear description of PRP components in studies, there is no consensus regarding the most appropriate PRP product to administer for each specific pathology. Research is warranted to determine best practices regarding the number of injections needed per pathology, the interval of multiple injections, dose-effect with platelet concentration, best practices for LR-PRP versus LP-PRP products, the need for exogenous platelet activation, and the use of local anesthetics which may reduce the pain associated with the procedure but also may have a negative effect on platelet function.

Autologous Conditioned Plasma (ACP)

ACP is a specific commercially available preparation of PRP, consisting of a single-spin LP-PRP injectate (ACP, Arthrex, Naples, Florida, USA). As this is an LP-PRP, it would be expected to have similar beneficial use as other LP-PRP preparations. There has been minimal evidence to support that ACP has specific use or benefit over other PRP preparations. In a study of ACP in knee OA, intra-articular injections of ACP had mixed results, suggesting possible benefit over HA.77 However, real-world data has not demonstrated clinically beneficial effects from ACP in pain or functional outcome scores.78,79 Overall, there is insufficient evidence supporting the use of ACP over other PRP preparations, or for its specific use for musculoskeletal conditions.

Leukocyte-Rich PRP

There is debate on the utility of including leukocytes in PRP preparations due to potential for secreted cytokines to cause inflammation. However, these preparations may also have potential in preventing infection and improving healing. The evidence to support the optimal leukocyte concentration in PRP formulations is controversial and has not been well-established in vivo. In tendon pathology, a laboratory study suggests that LR-PRP may be superior to LP-PRP on tenocyte proliferation.80 This is contrary to in vitro studies that suggest the opposite effect.81 A three-arm comparison of LR-PRP versus LP-PRP versus saline showed no difference in all three treatment groups in patellar tendinopathy, with all three cohorts demonstrating similar benefit at 1 year.82 This is consistent with a study of lateral epicondylitis, in which LR-PRP, LP-PRP, and saline cohorts had similar favorable outcomes at 8 weeks.83

Leukocyte-Poor PRP

Some in vitro studies have reported that a high leukocyte concentration within PRP could increase the expression of catabolic pro-inflammatory molecules, which could be detrimental to the joint, suggesting that LP-PRP may be better suited for OA.84,85 Some clinical data suggest that LR-PRP has a more positive effect on ligament and tendon pathologies, while LP-PRP has a more positive effect on joint OA pathologies.23,31,86–91

Overall, it is most likely that both LP-PRP and LR-PRP have favorable outcomes for OA. Authors in a meta-analysis suggest that exclusion of leukocytes in LP-PRP may be favorable for less local adverse reaction and pain following injection compared to LR-PRP.92 However, outcomes comparing LR-PRP and LP-PRP for intraarticular injection are similar, with both LR-PRP and LP-PRP showing similar clinically meaningful improvements in pain and function.92 In a direct comparison of LR-PRP versus LP-PRP in knee OA, an RCT of 192 patients who received three weekly injections of LR-PRP or LP-PRP showed similar outcomes of pain intensity and functionality at 12-month follow-up, suggesting that the presence of leukocytes did not significantly impact efficacy.93

To our knowledge, there have been no studies directly comparing LR-PRP to LP-PRP for intradiscal, epidural, or facet use. There is some belief that the LR-PRP may be the preferred injectate in intradiscal injections for possible anti-bacterial properties, however this has not been supported or tested in the literature.

In summary, there is some evidence supporting the role of leukocytes in growth factor release, and several studies showing that leukocyte-reduced, and often platelet-reduced, products may not be effective. The heterogeneity or non-classification of PRP in studies makes it challenging to assess the differential efficacy of platelet derivatives.

Platelet-Rich Fibrin

PRF is a PRP derivative where autologous platelets and leukocytes are present in a complex fibrin matrix. The fibrin matrix is thought to act as a scaffold to enable cell migration to a site of repair. This has led to its use particularly in tendon pathology, as the fibrin matrix may have beneficial effects in a tendon defect. This has largely been studied as an adjunct to arthroscopic surgical tendon repairs with limited evidence of support. An RCT studying rotator cuff tears compared arthroscopic surgery versus arthroscopic surgery with PRF adjunct and reported no significant between-group differences in pain intensity or structural integrity.94 This is concordant with a meta-analysis of 219 patients showed similar re-tear rates in arthroscopic repair with use of PRF for rotator cuff injury.95 To our knowledge, there have not been prospective studies of percutaneous injection of PRF for tendon pathology outside of a surgical setting.

Platelet Lysate

PL is a cell-free platelet derivative, in which the intracellular components of the platelet have been released and collected as an injectate. It has been studied as an injectate in intra-articular joints96 and tendons,97 consistent with other platelet-derived biologics with favorable benefit. PL carries a unique safety profile compared to other platelet derivatives as it is acellular and should theoretically not carry the potential risk for platelet aggregation or vascular occlusion, although this warrants further investigation. In a study of 470 patients treated for lumbar radiculopathy, PL injection mixed with local anesthetic and corticosteroid decreased pain and increased functionality up to 2 years post-injection.98

Consensus Point 2.There is insufficient evidence regarding the most appropriate PRP product (ACP, LP-PRP, LR-PRP, PRF, PL) to administer for each specific pathology. There is some evidence supporting the role of leukocytes in growth factor release, and several studies showing that leukocyte-reduced, and often platelet-reduced, products may not be effective. Further comparative research among these products is warranted, including details on dose-effect with platelet concentration and need for exogenous platelet activation (Level III, Grade I).

Question 3: What Injectable Solutions are used in Prolotherapy for the Treatment of Chronic Pain Conditions?

Prolotherapy is a form of regenerative therapy where non-biologic agents are used to stimulate inflammation and growth. Some non-biologic agents utilized in prolotherapy include hypertonic dextrose, phenol-glycerin-glucose (P2G), sodium morrhuate, and pumice.99–101 Hypertonic dextrose is a commonly used solution for prolotherapy. Hypertonic dextrose may be used in a concentration ranging between 10% and 25% and is often diluted in local anesthetics in a 1:1, 1:2, 1:3, 1:4, or 2:5 ratio before injection.100,101 Sodium morrhuate is less commonly used and may be considered as an alternative if hypertonic dextrose is ineffective. Sodium morrhuate is found in a 5% solution composed of 2% benzyl alcohol in sodium salts of cod liver oil. Sodium morrhuate is diluted to a 0.5% to 1% solution before injection.100 Pumice is another non-biologic agent, derived from lava rock and grounded in fine powder. Pumice permanently remains in the body after injection, and therefore, is used as a last resort when all other proliferative injections fail. Finally, although phenol-glycerin-glucose is no longer used in clinical settings,99 it is used in research settings. This agent is constituted of 25% dextrose, 25% glycerin, and 2.5% phenol and is often diluted in local anesthetic prior to injection.100

The underlying mechanism for all proliferative agents used in prolotherapy involves the stimulation of inflammation and tissue growth.99,101 Glucose transporter (GLUT) 1–4 are found on the surface of many cells and are responsible for the transport of glucose.101 The entry of high concentrations of glucose or irritation from other proliferative agents such as P2G and sodium morrhuate leads to cellular lysis and release of intracellular contents. This generates temporary low-grade inflammation and the production of cytokines.101 Subsequently, these cytokines act in a paracrine fashion on the surrounding cells to promote growth and healing.102 Fibroblasts and nerve cells are all activated upon exposure to proliferative agents.101 In addition to the inflammation caused by proliferative agents, the direct trauma from the needle disrupts the cellular membrane and blood supply. This leads to the release of inflammatory factors such as calcitonin gene-related peptide (CGRP), bradykinin, and prostaglandins that are implicated in healing.103 It is noteworthy to highlight the nascent nature of research on elucidating the mechanism of various proliferative agents in prolotherapy. More work is needed to clarify the mechanism of proliferative non-biologic agents and to develop well-defined protocols for their use in pain medicine.

Consensus Point 3.Prolotherapy has primarily been utilized in research settings. The most common agent utilized in prolotherapy is hypertonic dextrose with a proposed mechanism involving generation of temporary low-grade inflammation, followed by cytokine release that promotes growth and healing (Level II-2, Grade C).

Question 4: What is the Evidence for Treatment of Tendinopathies Using Injectable Biologics?

In the current and subsequent sections of the guideline, we summarize evidence for injectable biologics for specific painful indications. Study data are also summarized in Supplemental Tables 5–21.

Lateral Epicondylitis

Lateral epicondylitis (LE), or tennis elbow, describes an overuse injury of the extensor tendons attaching to the lateral humerus. While typically self-limited, injuries failing the initial healing response can lead to chronic, degenerative tendinopathy. Consequently, the focus of treatment may shift from anti-inflammatory medication to regenerative approaches. Corticosteroid injections (CSI) have traditionally been used in LE refractory to conservative management. While they can provide short-term symptomatic relief, their use is increasingly discouraged due to questionable efficacy, high rates of recurrence, and risk of further tissue damage.104 Dextrose prolotherapy has yielded outcomes comparable or superior to CSI and placebo.104 A meta-analysis of nine trials demonstrated positive outcomes with dextrose prolotherapy, although there was a moderate risk of bias.105

The use of autologous blood products has gained interest as an alternative intervention for recalcitrant LE. RCTs comparing either leukocyte-rich PRP (LR-PRP) or leukocyte-poor PRP (LP-PRP) to placebo demonstrated significant improvements in pain and function with both types over a year of follow-up.106,107 RCTs also reported superiority of PRP injections to CSI at 3 months to 2 years.108 Some trials reported that CSI may be equivalent or more effective for pain relief in the short-term, but this effect declined over time.109 Meta-analyses have reiterated this association: CSI demonstrates efficacy for 2 to 8 weeks, while PRP remains superior in the longer term (3 months to 2 years).110 PRP injections also yielded similar benefits to surgical intervention in multiple studies.111 However, they have not demonstrated efficacy as an adjuvant to surgery or tenotomy.112 Unlike PRP, autologous conditioned plasma (ACP) injections failed to show significant improvements versus placebo over 6 months in small RCTs.113 However, they were superior to CSI in a larger RCT with 1-year follow-up.114

Administration of BMAC was associated with symptom relief from 2 to 12 weeks in one uncontrolled trial.115 Two uncontrolled pilot studies reported progressive improvements in pain, performance, and anatomical findings noted on MRI over a year with A-MSCs.116,117 The use of these biologics deserves further investigation with high-quality RCTs.

Consensus Point 4. PRP injection for lateral epicondylitis is associated with superior long-term relief (3 months to 2 years) compared to corticosteroid injection (Level I, Grade B).

Consensus Point 5. Limited evidence suggests that BMAC and adipose-derived MSC injection for lateral epicondylitis may be associated with improvement in pain, physical function, and anatomical findings (Level II-3, Grade C).

Consensus Point 6. Intra-tendinous ACP injection is likely ineffective for improving pain control or functionality in lateral epicondylitis and other tendinopathies (Level I, Grade D).

Medial Epicondylitis

Medial epicondylitis (ME), also known as golfer’s elbow or little league elbow, is an overuse injury of the flexor-pronator tendon complex at the medial elbow.118 ME manifests as a result of progressive angiofibroblastic disorganization and tendinopathy from repetitive microtrauma. Partial or complete tears of the involved tendons and ligaments, such as the ulnar collateral ligament (UCL), may accompany the degenerative process, particularly in athletes. While conservative management may lead to symptom resolution in the majority of cases, interventional approaches for recalcitrant ME include injections, tenotomy, and surgery.

CSI is the only injection type studied in an RCT specifically targeting ME. In a double-blinded RCT of 58 patients with ME, methylprednisolone injections resulted in significantly more pain relief than saline at 6 weeks, but not at 3 or 12 months.118 This short-lived effect of CSI is consistent with data from other joint and tendon disorders. As with other locations, CSI use at the medial epicondyle is associated with lipoatrophy, depigmentation, and tendon damage.119

In relation to injectable biologics for ME, some evidence is present for PRP and ACS. A systematic review of retrospective studies concluded that either LR-PRP or LP-PRP led to comparable outcomes to surgery and tenotomy for pain relief and function.120 In cases of sport-related UCL tears, two uncontrolled prospective studies reported significant improvements with PRP in functional scoring and ultrasound findings at an average follow-up of 54 to 70 weeks, with an average return to play at 12 weeks.121,122 An uncontrolled study of 14 patients reported no significant benefit from the use of ACP over 6 months, although there was a high dropout rate.123

Consensus Point 7. PRP injection for medial epicondylitis may provide long-term relief and functionality compared to surgery and tenotomy (Level II-2, Grade C).

Achilles Tendinopathy

Achilles tendinopathy (AT) involves a painful degeneration of the triceps surae tendons. Biomechanical overload and tissue-level alterations lead to a progressive loss of matrix integrity. The Victorian Institute of Sports Assessment-Achilles (VISA-A) or Achilles Tendon Rupture Score (ATRS) are often utilized to assess the severity of tendinopathy. Management of AT may include activity modification, physical therapy, orthotics, and anti-inflammatory medications. The use of CSI has fallen out of favor due to lack of efficacy and increased risk of rupture.124 Complete tendon ruptures or partial tears that fail to improve with conservative management may warrant surgical repair. In both non-surgical and surgical cases, the use of injectable biologics has been explored as an adjunct to treatment.

Multiple case series and uncontrolled trials have reported promising clinical improvements with PRP injection for AT.125 However, these results were not reproduced in several RCTs comparing PRP injection to placebo.126 Repetitive meta-analyses of these RCTs also failed to demonstrate significant long-term differences in VISA-A scores, pain, or tendon parameters on imaging.127 Some meta-analyses did find a significant short-term improvement at 6 weeks in VISA-A in the PRP group.128 In the setting of Achilles rupture, there was no difference in ATRS or rate of re-rupture with PRP versus saline injections.129 As an adjuvant to surgical repair130 and endoscopic debridement,131 the addition of PRP failed to demonstrate meaningful clinical benefit.

The use of BMAC for both chronic AT and Achilles rupture has been reported by retrospective case series.132 SVF injections compared to PRP produced similar improvements on imaging in one RCT133 and superior scoring outcomes in the short-term in another,134 but neither study was placebo-controlled and imaging did not correlate with symptoms.

Consensus Point 8. There is inconsistent and conflicting evidence on the use of PRP and BMAC for treatment of Achilles tendinopathy. Similarly, the re-rupture rate was not different with PRP injection versus saline injection (Level I, Grade C).

Plantar Fasciitis

Plantar fasciitis (PF) presents with stiffness and pain near the medial tuberosity of the calcaneus that is typically worse when initiating walking. In the majority of cases, symptoms resolve with conservative management. For recalcitrant PF, local interventions such as extracorporeal shockwave therapy (ESWT) or injections have been utilized. CSI is commonly administered to manage severe pain associated with PF. However, several trials and meta-analyses have found them to offer similar analgesia to dry needling and inferior analgesia to alternatives such as ESWT and PRP.135 CSI has also been associated with an increased risk of plantar fascia rupture.135 Meta-analyses have demonstrated low-quality evidence supporting the superiority of dextrose prolotherapy to placebo, although there was moderate-quality evidence of its inferiority to other modalities such as CSI for PF.136 Similarly, a series of five HA injections alleviated PF pain more than placebo at 5 weeks in another RCT,137 although additional studies are warranted to confirm this effect.

Most RCTs generally support that PRP injections were superior to placebo and CSI for pain relief and functional outcomes over 6 to 24 months.138 Some RCTs with shorter follow-up and lack of sham controls reported comparable outcomes to CSI in the early study phase, which may be confounded by anesthetic or placebo effect.139 Despite the heterogeneity and inconsistent blinding in these studies, multiple meta-analyses have reiterated that PRP is superior to CSI by 6 months for PF.140

Two RCTs reported significant improvements in pain and function with injection of human amniotic/chorionic membrane (HACM) versus sham injections for PF up to 3 months,141 while a pilot RCT demonstrated comparable outcomes to CSI over 12 weeks.142 A meta-analysis comparing different injections for PF found HACM likely to be superior to placebo, although this warrants further study.143

Consensus Point 9. There is low-quality evidence supporting superiority of dextrose prolotherapy compared to placebo for plantar fasciitis, although moderate-quality evidence suggests that it is inferior to corticosteroid injections. Therefore, prolotherapy is not the first recommended injection option for plantar fasciitis (Level I, Grade C).

Consensus Point 10. There is consistent evidence supporting that PRP injections for plantar fasciitis are associated with superior analgesia and physical function compared to placebo and corticosteroid injections over the long-term (6 to 24 months), although outcomes may be comparable in the short-term (Level I, Grade B).

Patellar Tendinitis

There are few, low-quality studies investigating injectable biologics for patellar tendinitis.144 A review highlighted best practices in patellar tendinopathy treatment, suggesting that PRP may not provide additional benefit beyond conventional therapy.145 Studies suggest that this may be due to lack of consistent methodology on dose, frequency, and timing coupled with variable patient selection criteria.146 Two small studies report the use of MSCs in patellar tendinopathy. Pascual-Garrido et al treated eight patients and reported high patient satisfaction with the treatment at five years.147 Conversely, Rodas et al failed to demonstrate the superiority of MSCs compared to PRP treatment, although it should be noted that both arms had improvements in reported pain and function scores.148

Consensus Point 11. There is inconsistent and conflicting evidence from low-quality studies on the use of PRP and injectable biologics for the treatment of patellar tendinopathy. Our appraisal suggests that injectable biologics are not recommended as an adjunct to conventional therapy (Level II-3, Grade D).

Rotator Cuff Tendinopathy

With regard to the use of PRP to treat partial rotator cuff tears, several RCTs showed efficacy over CSI including one study that demonstrated that intralesional injections improved rotator cuff tear size.149–153 A systematic review examining the use of PRP for partial thickness rotator cuff tears consisted of eight studies on intra-articular, subacromial or intra-tendinous injection approaches. Although the review was inconclusive of the best injection approach, PRP injection was generally associated with functional improvement in rotator cuff tendinitis.154 A significant number of studies performed PRP injection as an adjunct to surgical repair, making the assessment of PRP effect challenging. Evaluation of certainty of evidence is difficult due to the heterogeneity of techniques (eg palpation-guided vs ultrasound-guided), location of injection (intratendinous/bursa vs intra-articular), and other variables. All clinical endpoints suggested that PRP is potentially superior to CSI for rotator cuff tendinitis. The three RCTs that showed no difference or some difference in only short-term outcomes used PRP preparations that were less than two times concentrated.155–157 This is important as Marx et al have suggested that the minimum definition of PRP is 4–5 times concentrated.158

The use of BMAC injection to treat partial rotator tears versus exercise has been investigated in an RCT with a published mid-term analysis showing efficacy and reduced tear size.157 A second study of BMAC demonstrated reduced tear size and efficacy when compared to exercise.159

Consensus Point 12. The use of PRP injection for rotator cuff tendinitis is likely to provide superior analgesia compared to corticosteroid injections, including intra-articular, subacromial, and intra-tendinous approaches. Studies with non-inferior outcomes utilized less than two times the platelet concentrations, highlighting the importance of