Biologic Treatment of Orthopaedic Injuries

EWI Symposium AAOS Research Symposium Updates and Consensus: Biologic Treatment of Orthopaedic Injuries

Abstract Robert F. LaPrade, MD, PhD Strategies that seek to enhance musculoskeletal tissue regeneration Jason L. Dragoo, MD and repair by modulating the biologic microenvironment at the site of injury have considerable therapeutic potential. Current and emerging Jason L. Koh, MD biologic approaches include the use of growth factors, platelet-rich Iain R. Murray, MD, PhD plasma, stem cell therapy, and scaffolds. The American Academy of Andrew G. Geeslin, MD Orthopaedic Surgeons hosted a research symposium in November Constance R. Chu, MD 2015 to review the current state-of-the-art biologic treatments of articular cartilage, muscle, tendon, and bone injuries and identify knowledge gaps related to these emerging treatments. This review outlines the findings of the symposium and summarizes the consensus reached on how best to advance research on biologic treatment of orthopaedic injuries.

iologic approaches to the treat- platelet-rich plasma (PRP), stem cell Bment of orthopaedic injuries therapy, augmentation of soft-tissue aim to optimize clinical outcomes healing, and the use of scaffolds. The by improving musculoskeletal tissue symposium participants identified healing. Despite considerable research fundamental changes in approach effort,1,2 biologic approaches have not that will be necessary to expedite yet achieved a sufficient evidence base clinical translation of biologic ther- to warrant widespread clinical appli- apies and recommended a frame- cation. In 2015, the American Acad- work for future research. emy of Orthopaedic Surgeons (AAOS) held a symposium to identify knowl- Systematic Approach to From the Steadman Philippon edge gaps related to biologic treatment Research Research Institute, Vail, CO of orthopaedic injury (BTOI) and to (Dr. LaPrade), Stanford University, establish some consensus on how Researchers have initiated 500 Redwood City, CA (Dr. Dragoo and . Dr. Chu), NorthShore University future research might be best directed. clinical trials evaluating mesenchy- HealthSystem, Chicago, IL (Dr. Koh), The experts who participated in the mal stem cells (MSCs) and .180 the University of Edinburgh, symposium were in universal agree- trials evaluating PRP (see clin- Edinburgh, United Kingdom ment that the most effective way to icaltrials.gov) in a wide range of (Dr. Murray), and Western Michigan University Homer Stryker M.D. School achieve meaningful translation of applications, demonstrating wide- of Medicine, Kalamazoo, MI therapies into clinical practice is to use spread interest in the advancement (Dr. Geeslin). asystematictranslational approach of biologic treatment approaches. J Am Acad Orthop Surg 2016;0:1-17 based on sound scientific evidence However, many of these studies were and greater integration between http://dx.doi.org/10.5435/ initiated without sufficient under- JAAOS-D-16-00086 investigators, regulators, and indus- standing of the disease process being try. Consensus was reached in the targeted or even of the attributes of Copyright 2016 by the American Academy of Orthopaedic Surgeons. areas of working with regulatory the biologic therapy being evaluated. bodies and industry, the use of Hurried translation of new therapies

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Copyright Ó the American Academy of Orthopaedic Surgeons. Unauthorized reproduction of this article is prohibited. AAOS Research Symposium Updates and Consensus: Biologic Treatment of Orthopaedic Injuries into clinical use presents an expected approach will facilitate progression bodies should be initiated early in the inefficiency as a result of the use of through the regulatory process.6 research process. poorly characterized and suboptimal In addition to being more system- preparations. If the results of these atic, future research into BTOI must Working With Regulatory studies do not demonstrate a clear better integrate academia, industry, Bodies benefit, potentially effective treat- and regulatory bodies. Academic ments may be dismissed as non- researchers, industry, and regula- A thorough discussion of BTOI effective, to the ultimate detriment of tory bodies all strive to develop safe requires an overview of the FDA patients. This disordered approach is and effective therapies for patients. regulatory process. It is important to in direct contrast with established AcentralgoaloftheAAOSsympo- differentiate regulation of biologics7 models of translational research that sium was to foster collaboration from regulation of medical devices.8 have been used successfully to deliver between academia, industry, and Combination products are classified effective therapies across multiple key regulatory parties, including the as drug/device, biologic/device, drug/ medical disciplines.3,4 FDA. The importance of establish- biologic, or a combination of all Fundamental to the translational ing channels of communication three types.7 or bench-to-bedside approach is a between parties through regular comprehensive understanding of meetings, dialogue, or online forums Human Cells, Tissues, and the disease process. This under- was identified. Strategies to foster a standing of the disease process Cellular and Tissue-based unified approach include improving facilitates the development of tar- Products standards of reporting, initiating geted therapies, which are evalu- The FDA regulates human cells, tis- new FDA pathways for the evalua- ated in the laboratory setting before sues, and cellular and tissue-based tion of biologic products, and the efficacy is studied in animal models products (HCT/Ps) as defined in Title and ultimately in patients.5 Simul- introduction of biologic treatment 21, Part 1271, of the Code of Federal taneously, clinicians provide feed- registries that provide real-time data Regulations.7 This category encom- back on clinical observations that on efficacy while facilitating early passes a wide variety of products that might stimulate further preclinical warning of potential adverse effects are intended for implantation, trans- research. This approach ensures to prevent harm to patients. plantation, infusion, or transfer into a that strategies are based on sound The consensus statements on gen- human recipient. Blood products scientific rationale and that inef- eral approaches to future research on and minimally manipulated bone fective therapies are identified and BTOI are therefore as follows: (1) marrow are not considered HCT/Ps. prevented from reaching time- biologics research should be based on HCT/Ps are categorized on the basis consuming and expensive animal a comprehensive understanding of of their level of risk and are further and clinical studies (Figure 1). the pathologic basis of disease and regulated under the Public Health Because regulatory frameworks adequate characterization of the Service Act.9 A three-tiered frame- favor potential therapies that are proposed biologic therapy, and (2) work of regulation has been extensively characterized, adop- where possible, industry partnerships described by Chirba et al.10 Category tion of a systematic translational and dialogue with relevant regulatory 1 products are considered to be low

Dr. LaPrade or an immediate family member has received royalties from Arthrex and Smith & Nephew; serves as a paid consultant to Arthrex, Össur, and Smith & Nephew; has received research or institutional support from Arthrex, ConMed Linvatec, Össur, and Smith & Nephew; and serves as a board member, owner, officer, or committee member of the American Orthopaedic Society for Sports Medicine, the Arthroscopy Association of North America, the European Society for Sports Traumatology, Knee Surgery and Arthroscopy, and the International Society of Arthroscopy, Knee Surgery and Orthopaedic Sports Medicine. Dr. Dragoo or an immediate family member serves as a paid consultant to or is an employee of Becton Dickinson, ConMed Linvatec, DePuy Synthes, Genzyme, Moximed, Össur, and RNL Bio; has received research or institutional support from ConMed Linvatec and Össur; has received nonincome support (such as equipment or services), commercially derived honoraria, or other non-research–related funding (such as paid travel) from EmCyte; and serves as a board member, owner, officer, or committee member of the American Orthopaedic Society for Sports Medicine. Dr. Koh or an immediate family member serves as a paid consultant to or is an employee of Aesculap/B. Braun Medical, Aperion Biologics, and Arthrex; has stock or stock options held in Aperion Biologics; and serves as a board member, owner, officer, or committee member of the American Orthopaedic Society for Sports Medicine, the Arthroscopy Association of North America, the Illinois Association of Orthopaedic Surgeons, and the Patellofemoral Foundation. Dr. Chu or an immediate family member serves as a board member, owner, officer, or committee member of the American Orthopaedic Society for Sports Medicine. Neither of the following authors nor any immediate family member has received anything of value from or has stock or stock options held in a commercial company or institution related directly or indirectly to the subject of this article: Dr. Murray and Dr. Geeslin.

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Copyright Ó the American Academy of Orthopaedic Surgeons. Unauthorized reproduction of this article is prohibited. Robert F. LaPrade, MD, PhD, et al

Figure 1

Schematic diagram demonstrating the pyramidal approach of translational medicine. In this bottom-up approach, preclinical understanding of the disease process facilitates identification and development of biologic therapies. Therapies optimized in vitro are studied in small-animal and large-animal models and finally in clinical studies of safety and efficacy.

risk and do not undergo HCT/P lated under section 351 must undergo Device Amendments specify general oversight.7 a process that includes communica- controls that apply to all three device Category 2 includes lower-risk tion with the FDA for completion of classes and pertain to adulteration, products (section 361) that meet an investigational new drug applica- misbranding, device registration and the following criteria: minimal tion, development of clinical trials, listing, premarket notification, banned manipulation, homologous use and a biologics license application.1 devices, notification and recall, prod- only, no combination products, and uct reporting, restricted devices, and lack of systemic effect.7 The primary Current Good Manufacturing Practice requirement for products regulated Medical Devices regulations. under section 361 is manufacturing The FDA regulates medical devices as Class I (eg, manual surgical instru- according to the Current Good class I, class II, or class III.8 Class ments) represents the lowest level of Tissue Practice regulations,11 which designation depends on the level of risk and regulatory control.13 In ensures that HCT/Ps do not contain risk to the patient and the extent of addition to the general controls communicable disease agents, are regulatory control determined nec- required for class I devices, class II not contaminated, and do not essary by the FDA to allow market- devices require additional special become contaminated.7 ing of the device. Since the passage of controls, including special labeling Category 3 includes higher-risk the Medical Device Amendments to requirements, performance stan- products (section 351) and is more the Food, Drug and Cosmetic Act in dards, postmarket surveillance, and heavily regulated. Products are 1976, new medical devices are FDA guidance documents. Examples placed into this category if they do placed into class III regulation unless include devices such as prosthetic not meet all four criteria for category a comparable device (predicate joints, implants for fracture fixation, 2. In addition to manufacturing ac- device) is available, and re- and interference screws for ligament cording to the Current Good Tissue classification may occur depending fixation. Class III devices require the Practice regulations, products regu- on assessment of risk.12 The Medical general controls and must undergo a

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Table 1 manner. The Japanese reform occurred through the passage of the Consensus Statements on Platelet-rich Plasma Pharmaceuticals and Medical Devices No. Statement Act and the Safety of Regenerative 1 An accepted nomenclature and classification system that Medicine Act. Dialogue with interna- encompasses autologous blood/plasma products and categorizes tional partners and collaboration preparations in sufficient detail is required to facilitate comparison among investigators, industry, and across studies. Efforts should be made to involve academics, regulators will be necessary to advance clinicians, and industry representatives in this process to encourage widespread adoption of the system. the field while ensuring the safety of patients. 2 The influence of donor variance and processing and delivery factors on the composition of PRP must be established. 3 A validated assay of the efficacy of PRP should be established for each clinical application. Platelet-rich Plasma 4 The relationship between PRP composition and efficacy should be established. Platelets contain growth factors that 5 Minimum standards of reporting for all studies (preclinical and stimulate the proliferation of local clinical) evaluating PRP must be established to facilitate progenitors, direct cell differentiation, communication and the interpretation and synthesis of scientific investigations. These standards must include measured and modify local inflammatory characteristics of the PRP and factors relating to the donor, responses.20 Knowledge of these processing, and delivery of the PRP. regenerative attributes has stimulated 6 Specific formulations of PRP should be matched with specific efforts to explore the use of autolo- pathologic indications. gous preparations rich in platelets for 7 Methods for establishing proof of safety and efficacy of PRP should the treatment of orthopaedic injuries, be determined. This process may require evidence of phenotype including tendon,21 muscle,22 carti- stability or viability for each indication. lage,23 and bone24 defects. These PRP = platelet-rich plasma preparations are made by obtaining blood from a patient and processing it to concentrate platelets above base- line levels. Some of these preparations premarket approval (PMA) process regulation already exists are ineligible contain leukocytes. The manipulation before marketing.14 Sheth et al15 for this process. of blood to obtain PRP is minimal, reviewed the process for obtaining facilitating the process of regulatory FDA approval for marketing of new approval. Initial results achieved in orthopaedic devices. Future Directions introductory in vitro studies were The 510(k) pathway to marketing is The BTOI symposium consensus promising25,26 but have not yet been used for most new medical devices and statement on working with regula- reflected in many clinical studies to requires comparison of the proposed tory bodies and industry is that part- date.27,28 The AAOS symposium device to a previously approved device nership of academia and industry participants identified several obsta- (ie, predicate device).16,17 If the new with regulators is necessary to mod- cles to the advancement of PRP device is found to be substantially ernize regulation of biologics and therapies and made recommenda- equivalent to the predicate device, the that insight should be obtained from tions on how future research efforts new device is classified into the same international models when applica- should be directed to overcome these class and is subject to the same ble. As outlined by Sheth et al15 and challenges. The consensus statements requirements. in an internal FDA review, critical on future research on PRP are sum- The FDA Safety and Innovation Act challenges facing the FDA and its marized in Table 1. was passed in 2012 and provides an mission include funding, staffing, Orthopaedic clinical researchers amended classification process known and necessary scientific expertise.19 have hurried to identify clinical as de novo.18 This process provides a Japan has recently undergone regu- applications of PRP on the basis of pathway to class I or II classification latory reform to support and accel- limited scientific rationale. Sympo- for products that have no legally erate research and development of sium participants stated that PRP marketed predicate device. Class III regenerative medicine and cell ther- research should be hypothesis driven devices, combination products, and apy and to expedite access to these and based on an understanding of the devices for which a classification treatments in a safe and effective specific pathology addressed by each

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Copyright Ó the American Academy of Orthopaedic Surgeons. Unauthorized reproduction of this article is prohibited. Robert F. LaPrade, MD, PhD, et al treatment method. Comprehensive Table 2 understanding of the disease pro- Variables That May Influence the Growth Factor Profile of Platelet-rich cesses may reveal the scenarios in Plasma which delivery of growth factors may be of therapeutic benefit. Variable Description Researchers have performed studies Donor Age of varying levels of evidence in efforts Gender to demonstrate the safety and benefi- Comorbidities Concurrent medications (including anti- cial effects of PRP in a broad range of inflammatories) applications, but the lack of standard- Nutritional status ization of PRP preparation method- Processing Blood collection and storage conditions ology and application procedures Spin protocol (speed, time) remains a substantial barrier. Collab- Activation protocol (agent, concentration, timing) orative development of standardized Storage assays or criteria to substantiate PRP Delivery Form of delivery (gel, solution) Timing of delivery in relation to isolation safety and efficacy may accelerate Timing of delivery in relation to activation regulatory pathways while minimizing Host factors (similar to donor factors) risk to patients. Injury chronicity

Nomenclature and high-density fibrin network, which incorporate all autologous blood/ Classification are also called leukocyte-poor plasma products. The nomenclature of PRP products is platelet-rich fibrin; and leukocyte- notoriously confusing and may hinder and platelet-rich fibrin (L-PRF) des- research efforts.29 Broadly speaking, ignates preparations that contain Standardization of the term PRP is used to describe leukocytes in a high-density fibrin Preparation autologous plasma formulations con- network.32 Mishra et al31 proposed PRP preparations and their growth taining a platelet concentration in a classification primarily targeting factor profiles are highly variable. excess of that found in peripheral sports medicine applications. Their Growth factor concentrations within blood. Other terms that have been system also describes four types of preparations may be influenced by used include platelet concentrates, PRP but is based on the presence of donor-specific factors and by pro- autologous growth factors, plasma leukocytes and activation or non- cessing and delivery factors (Table 2). rich in growth factors, platelet gel, and activation of the PRP, with addi- The blood concentrations of platelets platelet-rich fibrin matrix. tional subgroups based on the level and their contained growth factors, Although several authors have of platelet enrichment. The PAW leukocytes, and erythrocytes have attempted to formally categorize classification system of PRP is based large intersubject variation,34 and PRP,30-32 no classification has on the absolute number of platelets patient factors that contribute to this achieved widespread adoption, and (P), the manner in which platelet variability have not yet been well none describes PRP preparations in activation (A) occurs, and the pres- established. Numerous PRP formula- sufficient detail to enable compari- ence or absence of leukocytes (W).30 tions are used in experimental or son between studies. One classi- Current imprecision in terminology clinical research, with multiple possi- fication system categorizes PRP is confounded by emerging autolo- ble combinations of blood storage, formulations on the basis of the gous plasma products with low num- centrifuge spin protocols, and methods presence of leukocytes and the fibrin bers of platelets, termed platelet-poor of activation. Currently, .17 different architecture of the formulation: pure plasma.33 Awidelyaccepted,more commercial protocols are in use,35 platelet-rich plasma (P-PRP) refers to detailed classification system should each yielding products with different leukocyte-poor PRP; leukocyte- and speed the process of identifying the compositions and characteristics. The platelet-rich plasma (L-PRP) refers to optimal PRP preparation for each effects of PRP formulation on clinical PRP that contains leukocytes and indication and allowotherinvesti- efficacy are not well understood, and has a low-density fibrin network gators to replicate published data or protocols of application, such as the after activation; pure platelet-rich perform meta-analyses. The AAOS volume of PRP delivered and the fibrin (P-PRF) designates prepara- symposium participants stated that timing of the injections after injury tions without leukocytes and with a any new classification should or the commencement of symptoms,

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Copyright Ó the American Academy of Orthopaedic Surgeons. Unauthorized reproduction of this article is prohibited. AAOS Research Symposium Updates and Consensus: Biologic Treatment of Orthopaedic Injuries have yet to be determined. These fac- egy requires a full understanding of whereas that of fibroblast growth tors are not routinely included in PRP composition. A validated assay factor is 7.6 hours.50,51 The half-life the methods sections of research of PRP efficacy should be established of growth factors may be extended reports.36,37 The heterogeneity of PRP for each clinical application. when heparin is administered in con- preparation and application precludes The growth factor profile of PRP is junction with the therapy.51 Future the establishment of consistent and influenced by its cellular composi- studies evaluating cytokine-release successful protocols for therapeutic tion.42 An optimal concentration of kinetics according to activation pro- treatment. Future research must platelets has been proposed for bone cesses or methods of localization establish patient factors influencing regeneration (1,000,000 platelets/ within a tissue (such as a collagen PRP composition and identify the mm3),43 although no consensus has scaffold) are required for the proper optimal delivery system, delivery tim- been reached for other indications. application of PRP to address specific ing, concentration, and proteolytic The role of leukocytes, particularly clinical indications. condition (which limits the half-life of neutrophils, in PRP is controversial. many therapeutic molecules) of the Although some studies have dem- PRP. The establishment of a standard onstrated a positive role of leuko- Stem Cell Therapy reporting system for PRP is necessary cytes in PRP as anti-infectious and to facilitate communication, interpre- immune regulatory agents,44 other Stem cells are unspecialized cells that tation, and synthesis of scientific researchers have reported detrimen- have the ability to differentiate into investigations.38 tal results of leukocyte-rich prep- multiple different cell types or to arations in a wide range of replicate themselves. The ability of applications.45 The optimal concen- MSCs to differentiate into multiple Indication-specific trations of leukocytes, platelets, and musculoskeletal cell types, release Formulations erythrocytes for most applications regenerative growth factors, and Many of the growth factors and remain unclear. dampen immune responses holds cytokines present in PRP act within great promise for orthopaedic tissue opposing biologic pathways, with engineering. Despite their wide- some factors having beneficial effects Timing of Delivery spread application in clinical trials, in certain applications and deleterious The optimal timing of PRP delivery MSCs remain poorly understood, effects in others. Whereas trans- remains largely unexplored. PRP can and their use remains controversial.52 forming growth factor b1(TGF-b1) be delivered preoperatively, intra- Greater understanding of how MSCs may have beneficial profibrotic effects operatively, and/or postoperatively behave in their native environment is in tendon and ligament healing,39 it and with repeated dosing. Limited necessary so that these characteris- has been shown to be detrimental in studies to date suggest that the tics can be harnessed for the benefit the healing of muscle injury.40 Simi- expression of local factors is unique of orthopaedic patients.53 Key larly, the pro-angiogenic effects of to each tissue and pathology and challenges to the progress of MSC- vascular endothelial growth factor depends on the chronicity of the based therapies identified at the critical to muscle regeneration nega- injury.46,47 Each condition may have AAOS symposium include abstruse tively affect articular cartilage heal- a therapeutic window in which PRP nomenclature and definitions; regu- ing.41 Understanding the role of each would be of most benefit. The bio- latory ambiguity; lack of under- growth factor in the development of molecular environment at the site of standing of the native roles and specific disease processes will facilitate pathology should determine the mechanisms of MSCs; the paucity of the identification of therapeutically timing of PRP delivery. In the same assays to define efficacy of MSCs; relevant components of PRP for each way that growth factor profiles at a indefinite association between phe- indication and the development of site of injury evolve over time,46 the notypic characteristics and biologic customized PRP preparations most growth factors released from plate- functions; lack of consensus on the suited to the specific indication. This lets vary after activation.48 Growth most effective source of MSCs; lack customization could potentially be factors are released almost immedi- of establishment of standard prepa- achieved by means of manipulation ately after activation, and most have rations for specific indications; lack of processing variables or by the a short life span.49 For example, the of data on the long-term safety of removal or enrichment of certain biologic half-life of vascular endo- MSCs; ambiguity regarding the most growth factors, although the regula- thelial growth factor and platelet- effective methods of MSC delivery; tory implications of this approach are derived growth factor in circulating and lack of consensus on appropri- unknown. Any customization strat- blood is approximately 30 minutes, ate clinical study design.

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Copyright Ó the American Academy of Orthopaedic Surgeons. Unauthorized reproduction of this article is prohibited. Robert F. LaPrade, MD, PhD, et al

Stem cell therapies are the subject of accepted to be safe, as non- Attempts have been made to stan- regulatory ambiguity. As noted, homologous would notably delay dardize the nomenclature used in minimal manipulation is one of the clinical translation while sub- MSC research. However, variations criteria used by the FDA to define the stantially increasing costs. in methods of isolation, culture, and regulatory pathway that a biologic assay have resulted in complicated therapy must undergo. Cell prepara- and at times misleading nomencla- tions that are minimally manipu- Nomenclature and Definition ture. In 2006, the International Soci- lated, are for homologous use only, The principal types of stem cells are ety for Cellular Therapy produced are not combined with a drug or embryonic stem cells (ESCs), adult a position statement suggesting the device, and are autologous or used in stem cells, and so-called induced minimum criteria required to define a first-degree or second-degree blood pluripotent stem cells (iPSCs). ESCs MSCs.67 According to the statement, relative are regulated under section are pluripotent and are able to dif- MSCs must be plastic adherent and 361. In contrast, products regulated ferentiate into any adult cell type (ie, must express the cell surface antigens under section 351 include cell and all three germ layers). Their use is CD105, CD73, and CD90. Addi- tissue products that are cultured or ethically controversial and may be tionally, they must not express the more than minimally manipulated, associated with increased tumor- cell surface antigens CD45, CD34, are not intended for homologous use, ogenicity because of their broad dif- CD14, CD11b, CD79a, CD19, or are combined with a drug or device, ferentiation potential.57 Adult stem HLA-DR, and they must differenti- or are allogeneic. These products cells have more limited differentia- ate into osteoblasts, adipocytes, and require an investigational new drug tion potential, usually restricted to chondroblasts in vitro. However, exemption by the FDA and a bio- cells of a single germ layer. They these criteria have several limita- logics license application on file with reside within each organ system and tions. First, they are based on the FDA before clinical trials are ini- are responsible for maintaining attributes observed in the laboratory tiated, whereas products regulated tissue turnover by replacing cells environment and may not reflect under section 361 bypass much of lost through injury or aging.52 In characteristics of MSCs in their this regulation.54 2006, Takahashi and Yamanaka58 native environment.52 Furthermore, A major deficiency highlighted at described the successful dedifferen- multiple distinct populations of cells the AAOS symposium was the lack of tiation of mature skin fibroblasts have been found to fulfill these cri- strict definitions of minimal manip- back to cells with pluripotent teria.64-66 Future definitions of ulation and of homologous use, potential. These iPSCs can theoreti- MSCs must reflect their identity although examples of preparations cally differentiate into any cell type. in vivo and account for the hetero- not meeting such criteria are avail- The use of iPSC technology over- geneity of populations identified to able.55,56 For example, isolation of comes the ethical challenges associ- date. progenitor cells from adipose tissue ated with the use of ESCs, although by ex vivo enzymatic digestion has concerns regarding tumorogenicity been deemed not to meet the remain.59 Native Roles of Stem Cells requirement of minimal manipula- MSCs are adult stem cells that Despite the extensive use of MSCs in tion.55,56 However, some companies become the cells of the musculoskel- clinical trials to date, the contribution have been allowed to use enzymatic etal system, including bone, cartilage, of MSCs to regeneration in native digestion under section 351 regula- muscle, and ligament.53 Since the tissues is not well understood. tion. The response from FDA rep- publication of early descriptions of Although studies clearly demonstrate resentatives taking part in the MSC-like cells obtained from bone that MSCs have the capacity to per- symposium was that researchers marrow,60 cells with similar charac- form multiple functions (differentia- should engage with FDA officials teristics have been identified in adi- tion, immune modulation, and early in the process because each case pose tissue,61 muscle,61 skin,62 release of growth factors), the timing is evaluated on an individual basis. periosteum, blood,63 and other of and reasons for the adoption of Further guidance by the FDA is sources. These cells have been as- each phenotype are not clear. For required to clarify perceived ambi- signed multiple names, such as mul- MSCs to be fully exploited in thera- guity surrounding point-of-care cell tipotent adult progenitor cells,64 peutic use, the mechanisms that reg- therapies. Designating the use of marrow isolated multilineage induc- ulate their native behavior must be autologous, minimally manipulated ible cells,65 and multipotent adult understood. cells that are processed at the point stem cells.66 The relationship of these A key first step is to establish the of care, the use of which is generally cells to MSCs is currently unclear. native identity of the stem cells that

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Copyright Ó the American Academy of Orthopaedic Surgeons. Unauthorized reproduction of this article is prohibited. AAOS Research Symposium Updates and Consensus: Biologic Treatment of Orthopaedic Injuries contribute to tissue development and tomic origins has not been robustly cells present within any given organ. healing in musculoskeletal tissues. demonstrated. Despite fulfilling the Available MSC preparations vary in Researchers have recently established International Society for Cellular the degree to which MSCs are sepa- that perivascular MSCs contribute to Therapy criteria, MSC populations rated from other types of cells and in the regeneration of injured muscle68 from different sources or states the method by which this task is and bone.69 These findings required (native or cultivated) have shown accomplished. Whole bone marrow the use of sophisticated transgenic differences with respect to the im- cell suspensions and the stromal vas- mouse technology that labels cells munophenotype, secreted cytokine cular fraction of adipose tissue (the with fluorescent markers and allows profile, and results of proteome cellular component of lipoaspirate them to be traced as they differenti- analysis.75-77 Cloned human MSCs after removal of lipids) have been ate into mature tissues. This tech- isolated from fat default to adipo- used directly with the aim of har- nology should be used to identify genic potential, whereas those iso- nessing the potential of the contained populations of cells contributing to lated from bone marrow default to stem cells. However, both represent the regeneration of cartilage, me- osteogenic potential, suggesting that highly heterogeneous cell populations niscal tissue, tendon, and ligament. the tissue environment of origin that include inflammatory cells, Identification of progenitors con- influences the characteristics of hematopoietic cells, endothelial cells, tributing to tissue regeneration (and MSCs.75 nonviable cells, and other cell types.83 also contributing pathologically to the Studies comparing sources of MSCs Studies using these heterogeneous development of fibrotic tissue) will have been limited by a lack of stan- populations have demonstrated poor facilitate research into the mecha- dardization in the methods of prepa- and unreliable tissue formation84 or nisms governing the recruitment and ration of MSCs and in the outcome lower regenerative efficacy compared activation of MSCs. Key pathways measures used.78 The behavior of with cultured MSCs.85 Recent stud- that have been identified include Wnt MSCs is known to be influenced by ies have also suggested that the (osteogenesis, adipogenesis, chondro- individual differences in donors, presence of endothelial cells may genesis),70,71 TGF-b (chondrogenesis, method of isolation, duration in cul- inhibit bone differentiation and other fibrogenesis),72,73 and Sonic Hedge- ture, and culture conditions; therefore, lineages.86,87 hog (osteogenesis).74 appropriate comparisons can be made Laboratory culture has historically only if these factors are standard- been used to enrich and expand MSC ized.79,80 The development of robust populations.88 MSCs rapidly expand Sources of Mesenchymal and reproducible assays of the efficacy in laboratory conditions, over- Stem Cells of MSCs is urgently needed to allow growing other cell types (eg, hema- MSCs were first isolated from bone comparison of MSC populations in topoietic cells, endothelial cells) that marrow, and most studies to date these settings. are less suited to the conditions. A have evaluated cells from this source. Cellular and genetic comparison may detail that is often overlooked in this However, MSCs have been isolated provide considerable comparative process is the temporal change and from multiple organs and tissues, and insight and guide functional assess- convergent cell phenotype occurring the advantages and drawbacks of ments. Next-generation sequencing through the culture process that may each source have not been fully eval- allows the entire transcriptome of affect cell behavior and the thera- uated. In addition to intersource populations (and even that of single peutic efficacy of the preparation in variation, MSCs may differ between cells) to be analyzed and compared, and proportion to the number of cells it anatomic regions of the same tissue recent developments in single-cell array contains.76 type (eg, MSCs isolated from iliac platforms mean that heterogeneity More recently, advances in flow crest or calcaneal bone marrow, or within populations can now be cytometry have enabled MSCs to be MSCs isolated from adipose tissue in analyzed.81,82 purified to homogeneity in hours the abdomen, buttock, or in- without the requirement of cell cul- frapatellar fat pad) in terms of yield ture.89 Compared with the lower and characteristics. Understanding Preparation of Mesenchymal yield, limited donor sites, and mor- the specific attributes of cells from Stem Cells bidity associated with bone marrow each source might reveal populations The most effective methods of prepa- or periosteal harvest, adipose tissue of cells most suited to a given ration of MSCs in terms of purity and offers a well-documented, easily indication. modification in laboratory culture accessible, abundant, and dispens- The equivalency of MSC pop- have not yet been determined. MSCs able source of MSCs that can be ulations derived from distinct ana- represent only a small proportion of isolated using fluorescence-activated

8 Journal of the American Academy of Orthopaedic Surgeons

Copyright Ó the American Academy of Orthopaedic Surgeons. Unauthorized reproduction of this article is prohibited. Robert F. LaPrade, MD, PhD, et al cell sorting.90 Approximately 15 at the clonal level. More stringent with mixed results. Tendon-to-bone million perivascular MSCs can be studies using clonally derived MSC healing was a focus of the BTOI purified per 100 mL of lipoaspirate, populations may enable the identifica- symposium because of the known providing a sufficient number of cells tion of subpopulations of cells better biologic limitations of rotator cuff to treat a broad range of musculo- able to maintain their stem cell–like repair. Another area of focus was the skeletal disorders without culture properties. These potency assays could role of biologic augmentation in the expansion.90 The high purity and help increase the efficacy and safety of treatment of tendinopathy. potency of MSCs achieved in this cell therapies using large numbers of process will facilitate the demon- culture-expanded cells. Similarly, the Augmentation of Tendon-to- stration of product safety and effi- identification of subsets of MSCs iso- cacy required for regulatory lated through fluorescence-activated Bone Healing approval.6 The trade-offs of greater cell sorting may facilitate purification Tendon-to-bone healing is critical MSC numbers and unaltered and of populations most suited to specific for successful surgical treatment of heterogeneous cell preparations have indications. Ultimately, assays of effi- soft-tissue injuries. A clinically rele- not been sufficiently explored in the cacy or potency of MSCs must be vant model of tendon-to-bone heal- literature. indication specific. ing is surgical reattachment of the Methods of establishing proof of the rotator cuff to the humerus. The safety of stem cell therapy are needed. rotator cuff enthesis consists of dis- Further Research These methods must include identifi- tinct zones: tendon, unmineralized Because of the documented capacity cation of off-target and systemic effects fibrocartilage, mineralized fi- of MSCs to differentiate into mus- of stem cells delivered locally and the brocartilage, and lamellar bone.91 culoskeletal tissues, the authors of ability to definitively establish the risk Because surgical repair does not initial studies hypothesized that (currently considered minimal) of generate a normal enthesis, the risk MSCs would achieve a therapeutic malignant transformation. of re-tear is high, especially in effect by means of tissue engraft- Future studies must establish the patients with large rotator cuff ment. However, few studies have most appropriate method of deliv- tears.92 Some studies have demon- documented substantial beneficial ery of MSCs. The role of concomi- strated that improved radiographic effects of engraftment, although tant delivery of growth factors and appearance does not necessarily some benefit is likely to be achieved the response of MSCs to local sig- result in a better subjective out- through the trophic and immuno- nals should be further explored. come.93 Subjective outcome mea- modulatory properties of MSCs. Similarly, interactions between cells sures with better sensitivity may Further research is required to fully and carriers such as patches, scaf- improve correlation with imaging. characterize the mechanism by folds, and hydrogels must be estab- Biologic options for augmentation which MSCs exert beneficial effects. lished. Finally, appropriate controls of healing include growth factors and If engraftment is not central to the for clinical studies evaluating MSC- cell-based therapies. The use of mechanism of action, the need for based therapies must be clarified. growth factors to augment tendon actual delivery of cells could be Most clinical studies of articular healing has shown potential in ani- questioned. Considerable clinical cartilage repair have compared mal studies; however, growth factors hurdles (such as rejection and tu- MSCs with microfracture. The are not available for clinical use in morogenicity) and regulatory chal- AAOS symposium highlighted the isolated form.93,94 PRP serves as a lenges could be bypassed if the need for consensus on appropriate vehicle for application of a physio- responsible therapeutic cytokine control therapies for each indication logic mixture of growth factors to a released by cells were identified. to facilitate comparison between site of injury or surgical treatment, Whereas multiple rigorous assays are studies. The consensus statements although the clinical efficacy is the available to establish the function and on future stem cell therapy research subject of substantial debate. A maturation stage of distinct progenitor are summarized in Table 3. recent meta-analysis of randomized hematopoietic stem cells, stringent controlled trials by Zhao et al95 did assays to demonstrate the efficacy of not support the use of PRP in MSCs are lacking. Most researchers Augmentation of Soft- arthroscopic rotator cuff repairs. In have examined potentially heteroge- tissue Healing contrast, Warth et al96 reported a neous MSC populations derived from decreased re-tear rate in patients cultures with high plating density that Several strategies to augment soft- with large tears treated with double- do not accurately reflect cell behavior tissue healing have been attempted row repair and PRP. As noted,

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Table 3 been evaluated in animal studies. The BTOI consensus statements on aug- Consensus Statements on Stem Cell Therapy mentation of tendon-to-bone healing No. Statement are summarized in Table 4. 1 The progenitors contributing to tissue development, regeneration, and healing in each specific tissue must be identified. The mechanisms regulating this contribution must be characterized. Treatment of Tendinopathy 2 The optimum preparation of stem cells for each indication must be Clinically, tendinopathy is defined established in a systematic fashion. Considerations should include as the presence of pain and dys- cell number, concomitant use of growth factors, predifferentization, function together with histologic and vehicle. tendon pathology. The terminology 3 Mesenchymal stem cells isolated from different tissues must be compared to identify the most appropriate cell source for each is often confusing and inconsistent, specific indication. and the term tendinopathy has been 4 The mechanism responsible for therapeutic effects observed in recommended as an encompassing applications to date must be comprehensively characterized. descriptor for overuse-related 5 A standardized assay of stem cell efficacy is needed. clinical conditions.101 Acontin- 6 Methods for establishing proof of safety of stem cell therapy should uum of tendon pathology has been be determined in collaboration with industry and regulatory proposed, although correlation agencies. This process may require evidence of phenotype stability or viability. of clinical and histologic/imaging findings to facilitate treatment 7 The most appropriate control for clinical studies evaluating stem cell 102,103 therapy in each indication must be identified. selection is difficult. In a study of patients with acute tendon rupture, tendinopathy was present in 97% of patients; however, it also Table 4 was silently present in 34% of Consensus Statements on Augmentation of Tendon-to-Bone Healing control subjects, suggesting that histologic findings may not corre- No. Statement late with clinical symptoms.104 1 The stem cells and growth factors involved in tissue healing after injury Physical therapy is often the first- and repair of tendon to bone should be identified. line treatment of tendinopathy, and its 2 Animal models of tendon-bone injury that attempt to mimic the biology of use has been supported in controlled chronic tendon pathology in humans should be developed. studies.105-108 The authors of pre- 3 The optimal rehabilitation protocol for patients who have undergone clinical studies have evaluated the repair of tendon-bone injury should be established. potential role of PRP in the treatment 4 A combination of stem cells and growth factors is likely necessary to improve healing of rotator cuff tears. of tendinopathy by examining the effect of PRP on tendon stem cell 5 Improved imaging methods are needed to support correlation with 109 subjective and functional outcome measures. collagen production and inflammation.110 As noted, different formulations of PRP are likely to have different indications and treatment variability in the formulation of PRP demonstrated substantial improve- effects. Clinical results of the treat- and between patients may contribute ment in healing outcomes in patients ment of tendinopathy are mixed. to the discrepancies observed among treated with bone marrow aspirate Mishra et al111 published a random- in vitro studies, preclinical studies, concentrate containing MSCs com- ized controlled trial evaluating the and human randomized controlled pared with the control group (87% efficacy of leukocyte-enriched PRP in trials. versus 44% intact). Other strategies patients with chronic tennis elbow. As noted, manipulation of HCT/Ps to augment tendon-to-bone healing, The researchers found no differences results in FDA regulation under sec- including the use of recombinant between the patients treated with tion 351. However, clinicians have human parathyroid hormone,98 PRP and a control group treated with shown improved results without cul- doxycycline,99 gene therapy, perios- saline at 12 weeks, although at 24 ture expansion of stem cells. A land- teal flaps, growth factor–coated weeks the success rates for the PRP mark case-control clinical study with sutures and interference screws, and and saline groups were 83.9% and 10-year follow-up by Hernigou et al97 osteoconductive materials,100 have 68.3% (P = 0.037), respectively.

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112 Dragoo et al evaluated injection of Table 5 leukocyte-rich PRP compared with Consensus Statements on the Treatment of Tendinopathy dry needling in the treatment of refractory patellar tendinopathy; No. Statement both groups were also treated with 1 A uniform terminology for tendinopathy is required. Correlation of standardized eccentric exercises. The clinical and objective findings (histology and imaging) should be PRP group experienced significantly established to facilitate staging of tendinopathy. more clinical improvement compared 2 The pathophysiology of midsubstance versus insertional with the dry needling group at 12 tendinopathy must be further established. The timing and role of weeks but not at 26 weeks. In a inflammation and inflammatory mediators in initiation and progression of tendinopathy should be established. randomized controlled trial, de Vos 113 3 The role of tenocyte and tendon stem cells in tendinopathy should be et al evaluated the treatment of considered. chronic midportion Achilles tendi- 4 The optimal animal model of tendinopathy should be established. nopathy with eccentric exercises and This model should be supported by imaging that is sufficiently PRP or saline injection and found no sensitive to detect differences in clinical outcome. difference in pain, function, satisfac- 5 The role of mechanical stimulus in the initiation and healing of tion, or return to sports. tendinopathy should be investigated. Matrix metalloproteinases (MMPs) 6 Well-designed randomized controlled trials are required to evaluate treatment methods. are a family of zinc-dependent endopeptidases and include the collagenases found in the extracellular matrix. MMPs may play a role in tis- influence the interaction of cells with studies of autologous chondrocyte sue remodeling through the degrada- the scaffold. The use of scaffolds in transplantation.120 Determination of tion of released extracellular matrix the treatment of articular cartilage the optimal control treatment to be components. Tissue inhibitors of and osteochondral defects, meniscal used with each indication in studies metalloproteinases are native inhibi- pathology, and rotator cuff tears has of scaffold technology (ie, os- tors and bind to MMPs in the extra- been evaluated in preclinical studies, teochondral, meniscal, and rotator cellular space. An imbalance between in vitro studies, and some early cuff defects) is necessary and is MMPs and tissue inhibitors of metal- clinical studies. important from both scientific and loproteinases has been reported to Several institutions prospectively regulatory standpoints. The BTOI lead to excessive collagen break- study patients undergoing novel sur- symposium consensus statements on down.114 Inhibition of the inflam- gical procedures such as cartilage the use of scaffolds are summarized matory phase by blockage of MMPs repair.118 With industry financial in Table 6. has been proposed.115 Orchard support, development of a multi- et al116 reported successful treatment center registry will allow prospective of patellar tendinopathy with injec- evaluation of scaffolds and other Cartilage/Osteochondral tions of the MMP inhibitor aprotinin. similar implants, with a goal of Defects The BTOI consensus statements on improving patient safety. The regis- Safran et al121 outlined four pri- tendinopathy research are summa- try could be similar to the American mary materials used in cartilage rized in Table 5. Joint Arthroplasty Registry,119 a scaffolds: protein polymers (eg, multidisciplinary effort that involves collagen, fibrin), carbohydrate surgeons, medical device industry polymers (eg, hyaluronic acid, Scaffold Strategies for representatives, patient advocates, polylactic acid), synthetic/artificial Repair payers, and hospital administrators polymers, and composites of and has a primary mission of polymers. Safran et al121 identified Scaffolds can be categorized on the improving the care of patients who the optimal tissue-engineering basis of the presence or absence of undergo hip and knee arthroplasty. characteristics of cartilage scaf- cells,117 the synthetic or biologic To advance the use of scaffolds in folds (many of which may be also origin of the scaffold, mechanical the treatment of orthopaedic injuries, applied to other target tissue types) properties of the scaffold, and the high-quality evidence obtained as including biocompatibility, target location and tissue type rep- through randomized controlled trials noncytotoxicity, biodegradability, licated. The chemical composition will be necessary. Microfracture has cellular support, permeability, and microstructure of the scaffold been used as a control treatment in mechanical and chemical stability,

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Table 6 treated with meniscal allograft transplantation and reported 88% survival Consensus Statements on the Use of Scaffolds at 5 years, decreasing to 63% survival No. Statement at 10 years and 40% survival at 15 1 Because articular cartilage is a complex three-dimensional structure, years. 126 the use of spatially oriented scaffolds may improve biomechanical Di Matteo et al recently re- properties and cellular organization during incorporation. viewed preclinical animal studies 2 The ability to recruit cells into acellular scaffolds warrants further of meniscal scaffolds, including investigation. collagen-based meniscal implants. 3 The immune response to scaffolds and their breakdown products In a recent nonrandomized level II must be clarified in animal and human models. study by Zaffagnini et al,127 the use 4 Tissue-specific scaffolds should be designed to address the of a xenograft collagen meniscal biochemical and biomechanical roles of native tissue. implant (bovine Achilles tendon) 5 Long-term in vivo behavior of polymers must be characterized in a relevant model. for medial meniscal defects was 6 Improvement in the ability to quantitatively evaluate scaffolds after compared with partial medial implantation through noninvasive means (imaging) is necessary. meniscectomy. The implant demon- 7 Collaboration with researchers in other disciplines (eg, dentistry, strated promising results in terms of biomaterials) may be helpful in the selection of appropriate clinical outcomes and imaging. A materials for scaffolds. recent clinical study of 16 patients 8 The development of sufficiently sensitive clinical outcome measures treated with a polyurethane menis- will be essential for the evaluation of advanced treatment strategies. cus scaffold for segmental medial 9 For scientific and regulatory purposes, the appropriate control group meniscus defect demonstrated for comparison with scaffold treatment (eg, microfracture, sham surgery, hyaluronic acid injection, or rehabilitation) should be promising early clinical and func- clarified. tional improvements.128 10 Development of a registry, with industry financial support, to appropriately track scaffolds (and other bioactive implants) will be important both to ensure patient safety and to achieve regulatory Rotator Cuff Tears approval. A study by Kannus and Józsa104 re- vealed that most tendon ruptures are associated with preexisting degener- reproducibility, availability, and ver- TGF-b3group.Theabilityto ative lesions within the tendons. satility for use in the treatment of full- recruit cells into acellular scaffolds Furthermore, the number of MSCs thickness or partial-thickness lesions. warrants further investigation, in the greater tuberosity of patients In a recent systematic review of the including determination of the with a rotator cuff tear decreases as a use of scaffolds in the repair of artic- optimal chemoattractant or che- function of the time from onset to ular cartilage lesions, Filardo et al122 moattractants and scaffold treatment of the tear, resulting in compared single-stage and two-stage microstructure. increased tear size, chronicity, and procedures. Limited clinical data are degree of fatty infiltration.129 These available for the newer single-stage characteristics may be associated procedures. More recently, Kon Meniscal Defects with difficult repairs, and the use of et al117 evaluated the use of cells in Because of the morbidity associated scaffolds may be helpful. Biologic scaffold-based cartilage treatments. with meniscal defects, investigators scaffolds, typically animal or human In situ tissue regeneration in a single- continue to search for the optimal connective tissue grafts, have a stage procedure that does not require treatment to restore meniscal func- unique three-dimensional protein ex vivo cell expansion is an attractive tion. Methods that have been studied structure that may enhance interac- treatment option.123 Lee et al124 include allograft transplantation, tion with host tissue. However, tested this process using a three- cell-free scaffolds, and xenografts. biologic scaffolds have lower bio- dimensionally printed proximal Several authors have reviewed me- mechanical properties and less pre- humerus articular surface in rabbits niscal allograft transplantation and dictable degradation rates than do with or without TGF-b3 and found found that it provides a good solution synthetic grafts. A recent systematic improved functional (weight-bearing) in a difficult patient group. Noyes review did not demonstrate strong and histologic (hyaline cartilage and Barber-Westin125 recently re- evidence either for or against the use regeneration) outcomes in the viewed long-term results in patients of scaffolds.27

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The inability of surgical repair of implant to fill donor sites in patients Previous attempts to use synthetic rotator cuff tears to produce a normal undergoing osteochondral autograft materials for treatment of knee enthesis has been well documented transplantation.132 The authors of pathology, such as in anterior cruciate and was highlighted by Thangarajah the study reported incorporation of ligament reconstruction, have been et al,91 who recommended further bone in the autograft transplants, plagued by long-term complications studies to optimize indications for whereas the donor sites with syn- resulting from the breakdown of graft surgical repair. Smith et al92 outlined thetic plugs had evidence of fibrous material.135 Therefore, characteriza- attempts to use tissue engineering to tissue alone. On the basis of these tion of the biomechanical properties restore the normal enthesis for ten- findings, the authors of the study and biocompatibility of materials at don and ligament healing and recommended against the use of the time of implantation is not suffi- emphasized the importance of a this implant to fill osteochondral cient. The immune response to scaf- source of pluripotent cells and the defects. In a different study folds and their breakdown products, use of a scaffold with appropriate evaluating the characteristics of as well as the long-term behavior of biomechanical and biochemical this synthetic implant, cartilage- such polymers in vivo, must be clar- properties. Derwin et al130 evaluated sensitive MRI and quantitative T2- ified in appropriate animal models the biochemical and biomechanical mapping MRI were used assess the before these products are approved properties of several commercially morphologic characteristics and for human use. available scaffolds for rotator cuff incorporation of the implant.133 repair. They reported similar bio- The authors of the study reported chemical properties of the scaffolds that the appearance of the plug on Summary to tendon allograft; however, the imaging improved with increasing The use of biologic treatments that mechanical properties of the scaf- postoperative duration, and T2 contribute to a regenerative micro- fold, specifically elastic moduli, were relaxation times approached those environment has great potential to an order of magnitude lower than of normal articular cartilage. To improve healing rates and function in those of tendon allograft.130 The protect patient safety and evaluate patients with musculoskeletal injury. ability to attract and maintain resi- efficacy of scaffolds, the continued In efforts to speed clinical translation, dent cells is an important charac- use of noninvasive imaging to investigators have moved forward teristic of scaffolds. In an in vitro monitor scaffolds is recommended. with clinical trials evaluating biologic study, Beitzel et al131 found that Validation of imaging findings with therapies despite limited under- porcine collagen scaffolds had opti- appropriately sensitive outcome standing of the underlying pathologic mal adhesion and proliferation of measures is necessary. As imaging basis of disease and without full MSCs. technology improves and the use of characterization of the therapeutic scaffolds expands, the establish- agents. To reach the full potential of ment of consistent imaging proto- biologic therapies, future research Advanced Imaging for cols will be necessary. Monitoring of Scaffolds must return to a pyramid approach whereby clinical trials are inspired by Previously, surgeons used second-look comprehensive scientific under- arthroscopy to assess intra-articular Biocompatibility standing and supported with appro- surgical procedures. However, the Some authors have raised concerns priate animal studies. The common clinical trend toward the use of non- regarding the biocompatibility of aim of academia, industry, and reg- invasive advanced imaging affords scaffolds. Natural products such as ulatory bodies is to translate scientific investigators the ability to qualita- collagen derivatives, hyaluronic discoveries into safe and effective tively evaluate the status of implants acid, alginate, agarose, fibrin glue, therapies for patients. Greater com- and surrounding tissue. This litera- and chitosan have been reported munication and collaboration ture has largely focused on the integ- to have better biocompatibility.117 between these parties will accelerate rity of cartilage repair, including Algul et al134 evaluated a three- this translation. microfracture and autologous chon- layered scaffold consisting pre- drocyte implantation. More recently, dominantly of chitosan, alginate, and advanced imaging has been used to tricalcium phosphate for the treat- References evaluate bony incorporation with ment of osteochondral defects and scaffolds. reported noncytotoxicity according Evidence-based Medicine: Levels of In one study, CT was used to eval- to the International Organization for evidence are described in the table of uate the use of a synthetic multiphase Standardization standard. contents. In this article, references

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22, 36, 37, 105, 107, 108, and 111- (HCT/Ps). http://www.fda.gov/ 21. Andia I, Sanchez M, Maffulli N: Tendon downloads/biologicsbloodvaccines/guidance healing and platelet-rich plasma therapies. 113 are level I studies. References 23, complianceregulatoryinformation/guidance Expert Opin Biol Ther 2010;10(10): 95, 96, 127, and 129 are level II s/tissue/ucm091408.pdf. Accessed April 8, 1415-1426. 2016. studies. References 87, 97, 114, and 22. A Hamid MS, Mohamed Ali MR, 118 are level III studies. References 12. Jarow JP, Ahmed HU, Choyke PL, Yusof A, George J, Lee LP: Platelet-rich 28, 91, 116, 117, 120, 122, 125, Taneja SS, Scardino PT: Partial gland plasma injections for the treatment of ablation for prostate cancer: Report of a hamstring injuries: A randomized 128, 132, 133, and 135 are level IV Food and Drug Administration, American controlled trial. Am J Sports Med 2014;42 studies. Urological Association, and Society of (10):2410-2418. Urologic Oncology public workshop. 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