Techniques in Regional Anesthesia and Pain Management (2011) 15, 74-80

ELSEVIER

Techniques in

Regional Anesthesia& Pain Manas tt

Regenerative medicine in the fieLd of pain medicine:ProLotherapy, pLateLet-rich pLasma therapy, and stem celltherapy-Theory and evidence

David M. DeChellis, DO,a Megan Helen Cortazzo, MDa,b

From the "University of Pittsburgh Physical Medicine and Rehabilitation, Pittsburgh, Pennsylvania; and"Physical Medicine and Rehabilitation Outpatient Clinics; UPMC, Mercy-Southside lnterventional Spine and PainProgram; and University of Pittsburgh, Department of Physical Medicine and Rehabilitation, Pittsburgh, Pennsylvania.

The concept of "regenerative medicine" (RM) has been applied to musculoskeletal injuries dating backto the 1930s. Currently, RM is an umbrella term that has been used to encompass several therapies.namely prolotherapy, platelet-rich plasma therapy (PRP), and stem cell therapy, which are being usedto treat musculoskeletal injuries. Although the specific treatments share similar concepts, the mecha-nism behind their reparative properties differs. Recently, treatments that possess a regenerative qualityare resurfacing and expanding into the musculoskeletal field as potential therapeutic treatment modal-ities. RM, in the form of prolotherapy, was first used to treat tendon and ligament injuries. With theadvancement of technology, RM has expanded to PRP and stem cell therapy. The expansion of differentRM treatments has lead to its increase in the application for ligament and tendon injuries, muscledefects, as well as pain associated with osteoarthritis and degenerative disks. Recently, the use ofultrasound has been added to these therapies to guide the solution to the exact site of injury. We review3 forms of RM injection: prolotherapy, PRP therapy, and stem cell therapy.© 2011 Elsevier Inc. All rights reserved.

Historically the field of interventional pain medicine hasfocused primarily on spinal procedures. However, muscu-loskeletal injuries as a whole are the most common cause ofsevere long-term pain and often affect psychosocial status,social interactions, and employment. [ With a worldwideincidence of more than 100 million musculoskeletal injuriesannually, pain physicians have now begun to direct theirattention to this area.2 Regenerative medicine is a develop-ing field with the goal of inciting repair or replacement ofpathologic tissues through the augmentation of natural pro-cesses or bioengineered means? Included in this umbrella

Address reprint requests and correspondences Megan Helen Cor-tazzo, MD, Uni versity of Pittsburgh, Department of Physical Medicine andRehabilitation, 2000 Mary Street, 4th floor, Pittsburgh, PA 15203.

E-mail address: cortmh@upmc.edu.

1084-208X/$ -see front matter © 2011 Elsevier Inc. All rights reserved.doi: 1O.1053/j.trap.2011.05.002

of regenerative medicine are prolotherapy, platel.et-richplasma, and mesenchymal stem cell therapies. As increasingevidence on regenerative medicine is generated, more in-terventional pain physicians are looking to use this field forpain-mediated peripheral sources via percutaneous means."

Prolotherapy

The term "prolotherapy" first appeared in the medical liter-ature during the mid-19S0s and was described as a form oftreatment for "incompetent structures through the genera-tion of new cellular tissue. ,,5,6 Introducing an irritating. sub-stance to induce healing has been used since the time ofHippocrates 7

; however, the modern use of prolotherapy inmusculoskeletal injuries can be traced back to the 1930s.8.9

DeChellis and Cortazzo Regenerative Medicine in the Field of Pain Medicine 75

Proliferant solutions used in this form of treatment arehypothesized to induce collagen deposition through chemo-modulation, as well as the "temporary neurolysis" of pe-ripheral nociceptors through chemoneuromodulation. Mod-ulation in this setting is purported to be mediated bymultiple growth factors and cytokines.6,10-12 To date, theexact mechanism of prolotherapy remains elusive and hasnot been well defined by high-level evidence studies.P

Although the exact proliferant used in prolotherapy oftenvaries, all solutions, excluding chemotactic agents, have theuniversal effect of inciting local tissue irritation, whichleads to an influx of inflammatory cells. Inflammation, be-ing the first step in the wound-healing cascade, results in theend-product of fibroblast proliferation with the subsequentdeposition of collagen.!" The different proliferants can beclassified into 3 classes. Osmotic agents, which includehyperosmolar dextrose, zinc sulfate, and glycerin, act bydehydrating local cells to the point of rupture in a processreferred to as "osmotic shock.,,14.15Phenol, guaiacol, andpumic acid belong to the second class known as irritants andact by either directly damaging cell membranes or causinglocal cells to become antigenic.l''i'" Chemotactic agents arethe final class and include the commonly used sodiummorrhuate. This class is purported to be a direct chemotacticagent to inflammatory cells, which differs from the formerclasses that induce inflammation indirectly. 14The injectionprotocols of these proliferants lack standardization and dif-fer with regard to volumes and concentration of proliferantsused, frequency of injections, injection technique, and con-current cointerventions.V''"

Now more commonly referred to as "regenerative injec-tion therapy" (RIT), prolotherapy is currently used in a widevariety of chronic pain entities. The effects on tendons andligaments have been widely discussed in medical literature,ranging from animal studies to randomized controlled trialsin humans. Animal studies have demonstrated Rl'I''s signif-icant advantageous effect on tendon cross-sectional area'"and strength.F" This is in contrast to animal studies regard-ing ligaments that have been separated by discord. WhileLiu and colleagues found an increase in force failure, mass,and fiber diameter in ligaments." a more recent studyshowed RIT had no significant effect on fiber diameter orforce failure.22

In human trials, Reeves and Hassanein showed signifi-cantly improved ligamentous stability and knee flexion inpatients with anterior cruciate ligament laxity 36 monthsafter hyperosmolar dextrose injections.P Case reports havealso demonstrated magnetic resonance imaging verified re-pair of complete anterior cruciate ligament and Achillestendon tears following a series of RIT, without surgicalintervention?4,25 In addition, RIT has been documented asa favorable treatment modality in overuse syndromes suchas lateral epicondyle tendinopathy, Achilles tendinosis, coc-cygodynia, chronic groin pain, and plantar fasciitis?6-32 Thebenefits of intra-articular RIT have been assessed in multi-ple joints. Reeves and Hassanein have reported significantreduction in pain, as well as radiologic improvement, in

patients with knee osteoarthritis (OA).33 Patients with fingerOA also showed significant improvements in pain and rangeof motion following hyperosmolar dextrose injections com-pared to controls at 6 months. II A randomized controlledtrial of the sacroiliac joint demonstrated significant im-provement in pain after intra-articular RIT. Although thepain was not statistically significant in comparison withintra-articular steroid injection, the RIT group had a longerperiod of pain reliefr'"

Studies for the use of RIT in neck and back pain havebeen widely investigated. Its use for cervical-mediated painhas been promoted, with retrospective studies demonstrat-ing improvement in pain and function following whiplashinjuries.35.36High-level evidence regarding nonspecific lowback pain has been less congruent.15.16.37-41Complicated bylimitations in methodology and the heterogeneity of clinicalprotocols, studies of RIT on low back pain have beendifficult to interpret collectively.41.42 However, the responseof leg pain secondary to moderate-to-severe lumbar .degen-erative disk disease appears promising in the case series byMiller and colleagues. This uncontrolled study showed sta-tistically significant pain improvements in patients treatedwith 25% dextrose, suggesting RIT may have an indicationin this specific patient population.P

Despite a minimal volume of robust evidence to support.its use in musculoskeletal pathologies, RIT's overall safetyprofile and strong anecdotal evidence leads many physiciansto continue using this treatment modality for a wide array ofchronic pain entities. 13.44

Platelet-rich plasma therapy

In the search for better modalities in the nonsurgical treat-ment of musculoskeletal injuries, platelet-rich plasma ther-apy (PRP) has recently been thrust to the forefront of publicattention.45.46 Initially used clinically in the fields of car-diothoracic and maxillofacial surgery in the late 1980s andearly 1990s, PRP has since been adopted into the field ofmusculoskeletal medicine.47-49 The concept behind its useas a nonsurgical treatment modality is to place it directlyinto areas of soft tissue pathology, via percutaneous injec-tion, to facilitate tissue regeneration. Healing is theorized tooccur secondary to the PRP's ability to augment the recruit-ment, proliferation, and differentiation of cells involved inthe regeneration of injured tissue.48-51 These actions arepurported to be mediated by numerous growth factors andbioactive proteins secreted by PRP's platelets followingactivation, in a process known as degranulation.Y" Theexact mechanism by which PRP acts to augment the endog-enous healing process remains elusive despite extensiveresearch.

PRP by its strictest definition is an autologous sample ofblood with a concentration of platelets above the physio-logical baseline.49.52 Following the extraction of autologousvenous blood with a large-gauge needle to prevent prema-

76 Techniques in Regional Anesthesia and Pain Management, VollS, No 2, April 2Qll

ture platelet activation,53 platelets are separated from otherblood components and further concentrated. 50 This occursthrough a centrifuge process, in which platelets are able tobe isolated from the other cell components of blood basedon their physiological size.50 The further concentrating ofplatelets occurs with subsequent centrifuge cycles." Al-though some authors suggest a minimal platelet concentra-tion to be "therapeutic;>4&.52,55studies with lesser concen-trations have shown good results.52.56. Numerous PRPpreparation centrifuges are available for clinical use, witheach system producing a slightly variable end product Differ-ences include platelet concentration, presence of leukocytes,and total amount of PRP produced.49.so.57The administrationprotocol of PRP currently remains nonstandardized. Althoughhistorically "activated" by exogenous substances, evidencemay now suggest that PRP may be activated endogenously bytype I collagen in vivo.58 This degranulation method mayreduce the loss of platelet-secreted bioactive mediators and

. also the risk of activator agent-induced complications suchas coagulopathy .4.50.5J

The effects of PRP have been extensively documentedfor a variety of tissue types, in a vast number of differentlaboratory and clinical settings. Despite the uncertainty ofits intrinsic healing mechanism and administration protocoldifferences, the early evidence of PRP's use for musculo-skeletal-mediated pain entities has been encouraging. Invitro and animals studies have demonstrated positive resultsin numerous types of tissue injuries that frequently plaguepatients including muscle.i" tendon,60,61 meniscus,62 liga-ment,63 cartilage,64-66 osteochondral surfaces." nerve,68.69and intervertebral disks.70,71 Although anecdotal evidenceexists to support its percutaneous clinical administration formuscle72-74 and acute ligamentous pathology,50,75 high-level evidence is mostly void. Pain generated from connec-tive.tissue of the foot has had minimal investigation. A pilotstudy of plantar fasciitis reported nearly 80% of patientsshowed improvement at 1year following ultrasound-guidedPRP. injections. 76

OA occurs in up to 9.6% of men and 18% of women over60 years of age and is expected to be the fourth leadingcause of disability within the next 10 years. 1 Local anes-thetic and corticosteroids historically used for treatment arenow being called into question, due to their purported det-rimental effects on structure and inability to affect the nat-ural history of OA.77-81 The use of PRP as a treatmentmodality has gained recent interest and may be 1possiblefuture candidate to treat this condition. Two recent pilotstudies, including 1 involving over 100 degenerative knees,showed data suggestive of pain reduction, improved kneefunction, and improvement in quality of life at 1 year fol-lowing intra-articular PRP injection.82.83 Although long-term follow-up demonstrated a decline in pain control, painimprovement remained above baseline."

The use of PRP for chronic tendinopathies has beeninvestigated, with pilot studies showing advantageous ef-fects stemming from its percutaneous administration at var-ious anatomical locations.85-87 Perhaps 1of the most com-

pelling pieces of literature to support the use of thistreatment modality in tendinopathy has been described in alevel 1 evidence study by Peerbooms and colleagues. Theauthors evaluated the response to intratendinous injectionsof either PRP or corticosteroids in patients with refractorylateral epicondyle tendinopatby. The results showed a sig-nificant difference in pain control and function in favor ofthe PRP group at 26- and 52-week follow-ups.88

Much like RIT, the overlying theme that precludes strongcomparison of studies in the current medical literature is thelack of randomized controlled trials with protocol standard-ization. Future studies need to address questions regardingPRP, such as possible long-term side effects associated withadministration, how storage of PRP affects efficacy, andhow the presence of leukocytes effects different tissue sub-types. Procedural standardization also needs to be addressedin terms of injection technique, postinjection protocol, andconcurrent use of nonsteroidal anti-inflammatory drugs .

Stem cell therapy

Adult tissues often have the ability to repair and regeneratefollowing injury. To date, the exact mechanism of repair ispoorly understood; I however, it is hypothesized to occurthrough the proliferation and differentiation of cells thatultimately restore == functionality. One possible expla-nation is found within nonhemopoietic progenitor cellsfound in pathologic ltissue. as well as cell reservoirs at otherlocations, which may help to provide this reparative capa-bility.89 These regenerative-capable cells are commonly re-ferred to as mesenchymal stem cells (MSCs), or bone marrowstromal cellS.89,9OMSCs are pmported to be multipotent.cells,with the capability of replicating as undifferentiated.cells" andalso multilineage differentiation in response to local cellsignaling pathways.89,9O,92-95

Over 40 years ago, bone marrow was the first loc.ationfrom which stem cells were isolated.92•95,96 Since that time,stem cells have been found in a variety of tissue typesincluding adipose97-99and synovium, among others.4,97.100Differences in marker gene expression between marrow-derived MSCs and MSCs from other sources have beendemonstrated. However the effects of these differences havenot been fully delineated.94,101 Conflicting evidence cur-rently exists regarding the differentiation efficacy of MSCsobtained from different stem cell reservoirs, such as marrowand adipose tissue.101,102In addition, there is controversysurrounding the quality of stem cells derived from bonemarrow or adipose MSCs with respect to growth kinetics,cell senescence, and multilineage differentiation poten-tial.10l.l02 These differences in MSCs and mesenchymaI-like cells have prompted the International Society for Cel-lular Therapy to create minimum criteria for MSCs based onsurface marker expression. activity in culture, as well asdifferentiation potential. 103

Evidence suggests that in response to injury, MSCs arefirst mobilized from perivascular niches into peripheral.cir-

DeChellis and Cortazzo Regenerative Medicine in the Field of Pain Medicine 77

culation,90,I04-I06 with subsequent migration into damagedtissues,90 Although the exact reparative mechanism ofMSCs is uncertain, early evidence suggested that these cellsmay differentiate into native cells of the injured tissue. Morerecent evidence suggests MSCs may also induce healingthrough a paracrine-like effect. This includes the mobiliza-tion of MSCs to specific injured tissues, followed by theability for these reparative cells to secrete bioactive factorsthat induce a regenerative microenvironment known as"trophic activity.,,9o,lo7

Based on their augmenting effects, physicians are nowharvesting these cells for clinical application. Following theaspiration from autologous bone marrow, MSCs are typi-cally isolated from other marrow cells and concentrated in aseries of steps. The process of isolating MSCs, which are asmall portion of marrow's nucleated cell fraction, occursmost often through their adhesion to plastic in tissue cul-ture." Evidence by Hemigou and colleagues has suggestedthat a minimum number of progenitor cells per volume isneeded in order for percutaneous injections to be efficaciousin tissue repair.108 Since bone marrow aspiration containsan MSC concentration well below this suggested "mini-mum," the isolated MSCs are further cultured for multiplepassages to induce cell expansion." A passage includes theremoval of the isolated cells from culture, with replacementinto culture media with new nutrients and growth factors forfurther cell expansion. This total process typically takesseveral weeks from the time of aspiration until the finalproduct is administered to the patient.4,109

Literature shows acute skeletal muscle injury in humansleads to the mobilization of MSCs into vascular circula-tion.IOSFurther studies have demonstrated the stepwise pro-gression of bone marrow-derived MSCs into eventual multi-nucleated muscle fibers.v'" In the use of tendons,intratendinous therapy induces histologic and biomechani-cal improvements in the early stages of tendon healingfollowing injury. III Recent evidence by Mirsha and col-leagues also suggests other regenerative medicine modali-ties such as PRP may enhance the effect of MSCs. Theauthors of this study showed a statistically significant en-hancement in MSC proliferation when exposed to PRP invitro compared to controls. I12

MSCs' effect on cartilage defects and OA has been wellstudied and reported. Their ability to differentiate into chon-drocytes has been well documented, with evidence suggest-ing that the use of dexamethasone in micro doses may directdifferentiation toward a chondrogenic lineage." Full-thick-ness cartilage tear repairs are purported to occur solelythrough the actions of MSCs. Il3 However, MSCs fromosteoarthritic marrow are found to have reduced prolifera-tion rates, reduced chondrogenic activity, and an increasepredisposition towards osteogenic differentiation. I I4, 115

These limitations to repair have led clinicians to search fora treatment modality to augment the healing of cartilagethrough percutaneous intervention. Animal studies haveshown evidence of both morphologic and histologic im-provements in cartilage healing, as well as meniscus regen-

eration, following the treatment of intra-articular injectedMSCs suspended in hyaluronic acid. 116,1I7 Percutaneousinjection of bone marrow-derived MSCs in a patient withknee OA yielded a 95% reduction in pain and magneticresonance imaging evidence of increased meniscus and car-tilage volume."

A surge of interest has been noted in recent medicalliterature regarding the use of regenerative medicine tocombat intervertebral degenerative disk disease. Cells ex-pressing stem cell markers have been identified in interver-tebral disksYs MSCs have been 1 of the primary targets ofcell therapy in the treatment of disk degeneration, withpositive effects seen in animal and in vitro studies. MSCshave shown a superior ability to produce extracellular ma-trix compared to more terminally differentiated cells. Othereffects of MSCs include the ability to differentiate intodisk-like cells, prevent disk cell apoptosis, limit disk cellsenescence, and retard the local immune system. Theoreti-cally these effects should play a role in preventing theoverall degenerative process of intervertebral disks. I 19 Theclinical use of MSCs for degenerative disk disease is scantin current literature; however, a recent case study demon-strated significant pain reduction following the percutane-ous placement of MSCs into lumbar degenerative interver-tebral disks.109 This evidence may suggest further clinicaltrials are needed to determine the role of these cells as afuture treatment mobility for intervertebral degenerativedisk disease.

Although increasing data are being generated to supportthe use of MSCs in musculoskeletal injuries, most studies tothis point have been conducted using in vitro and .animalmodels. As some authors have appropriately suggested, theeffects of MSCs may differ in clinical use given the phys-iological differences in cellular composition and me.chanicalloading within species;'?" Many questions and unverifiedtheories still remain regarding the clinical production, use,and placement of these naturally occurring regenerativecells.

Conclusions

Regenerative medicine is a new intriguing concept on thehorizon in the field of pain medicine. Although continuedresearch is greatly needed to determine efficacy and safetyprofiles, early evidence may foreshadow its future use inclinical practice.

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