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Duchenne muscular dystrophy (DMD) is a progressive muscle disease, characterized by mutations in the X-linked dystrophin, that has several therapeutic options but no curative treatment. Transplantation of muscle progenitor cells for treatment of DMD has been widely investigated; however, its application is hindered by limited cell survival due to the harmful dystrophic microenvironment. An alternative approach to utilize progenitor cells and circulatory factors and to improve the dystrophic muscle pathology and microenvironment is through parabiotic pairing, where mice are surgically sutured to create a joint circulatory system. Parabiotic mice were generated by surgically joining wild type (WT) mice expressing green fluorescent protein (GFP) with mdx mice. These mice developed a common circulation (approximately 50% green cells in the blood of mdx mice) 2-weeks after parabiotic pairing. We observed significantly improved dystrophic muscle pathology, including decreased inflammation, necrotic fibers and fibrosis in heterogenetic parabionts. Importantly, the GFP + cells isolated from the mdx mice (paired with GFP mice) underwent myogenic differentiation in vitro and expressed markers of mesenchymal stem cells and macrophages, which may potentially be involved in the improvement of dystrophic muscle pathology. These observations suggest that changing the dystrophic microenvironment can be a new approach to treat DMD.

Background:Platelet-rich plasma (PRP) and bone marrow concentrate (BMC) are orthobiologic therapies with numerous growth factors and other bioactive molecules. Before the clinical utility of PRP and BMC is optimized as a combined therapy or monotherapy, an improved understanding of the components and respective concentrations is necessary.Purpose:To prospectively measure and compare anabolic, anti-inflammatory, and proinflammatory growth factors, cytokines, and chemokines in bone marrow aspirate (BMA), BMC, whole blood, leukocyte-poor PRP (LP-PRP), and leukocyte-rich PRP (LR-PRP) from samples collected and processed concurrently on the same day from patients presenting for elective knee surgery.Study Design:Cross-sectional study; Level of evidence, 3.Methods:Patients presenting for elective knee surgery were prospectively enrolled over a 3-week period. Whole blood from peripheral venous draw and BMA from the posterior iliac crest were immediately processed via centrifugation and manual extraction methods to prepare LR-PRP, LP-PRP, and BMC samples, respectively. BMA, BMC, whole blood, LR-PRP, and LP-PRP samples were immediately assayed and analyzed to measure protein concentrations.Results:BMC had a significantly higher interleukin 1 receptor antagonist (IL-1Ra) concentration than all other preparations (all P < .0009). LR-PRP also had a significantly higher IL-1Ra concentration than LP-PRP ( P = .0006). There were no significant differences in IL-1Ra concentration based on age, sex, body mass index, or chronicity of injury in all preparations. LR-PRP had significantly higher concentrations of platelet-derived growth factor AA (PDGF-AA) and PDGF-AB/BB than all other preparations (all P < .0006). LR-PRP also had significantly higher concentrations of matrix metalloproteinase 1 (MMP-1) and soluble CD40 ligand than all other preparations (all P < .004). LP-PRP had significantly higher concentrations of MMPs, namely MMP-2, MMP-3, and MMP-12, than all other preparations (all P < .007).Conclusion:BMC is a clinically relevant source of anti-inflammatory biologic therapy that may be more effective in treating osteoarthritis and for use as an intra-articular biologic source for augmented healing in the postsurgical inflammatory and healing phases, owing to its significantly higher concentration of IL-1Ra as compared with LR-PRP and LP-PRP. Additionally, LR-PRP had a significantly higher concentration of IL-1Ra than LP-PRP. In cases where increased vascularity and healing are desired for pathological or injured tissues, including muscle and tendon, LR-PRP may be optimal given its higher overall concentrations of PDGF, TGF-β, EGF, VEGF, and soluble CD40 ligand.

PURPOSE To quantify and compare normative catabolic and anabolic factor concentrations in leukocyte-rich platelet-rich plasma (LR-PRP) at various time points, including baseline, 1 week after initiating naproxen use, and after a 1-week washout period. METHODS Asymptomatic healthy donors aged between 18 and 70 years were recruited (average age, 36.6 years; range, 25-64 years). Subjects were excluded from the study if they were actively taking any prescribed medications or nonsteroidal anti-inflammatory drugs (NSAIDs) or if they had any of the following at present or previously: blood or immunosuppression disorders, cancer, osteonecrosis, rheumatoid arthritis, avascular necrosis, NSAID intolerance, gastrointestinal or peptic ulcer disease, or kidney dysfunction. The anabolic factors vascular endothelial growth factor, fibroblast growth factor 2, platelet-derived growth factor AB (PDGF-AB), and platelet-derived growth factor AA (PDGF-AA) and the catabolic factors interleukin (IL) 1β, IL-6, IL-8, and tumor necrosis factor α in LR-PRP were measured. Peripheral blood was drawn at 3 time points: baseline, after 1 week of naproxen use, and after a 1-week washout period. RESULTS The angiogenic factors PDGF-AA (44% decrease in median) and PDGF-AB (47% decrease) significantly declined from baseline (P < .05) after 1 week of naproxen use. There was a significant recovery (P < .05) of PDGF-AA (94% increase) and PDGF-AB (153% increase) levels after the 1-week washout period, with a return to baseline levels. The catabolic factor IL-6 also had a significant decline from baseline (77% decrease in median, P < .05) after 1 week of naproxen use. After a 1-week washout period, the IL-6 level was similar to the baseline level (130% increase, P < .05). CONCLUSIONS Naproxen use diminished several biological factors in LR-PRP; however, a 1-week washout period was sufficient for the recovery of PDGF-AA, PDGF-AB, and IL-6 to return to baseline levels. Tumor necrosis factor α, IL-1β, IL-8, vascular endothelial growth factor, and fibroblast growth factor 2 did not show differences between the 3 time points of data collection. Discontinuing NSAIDs for a minimum of 1 week before LR-PRP treatment may improve certain biological factor levels. LEVEL OF EVIDENCE Level II, prospective comparative study.

Background:Osteoarthritis (OA) is one of the leading causes of disability in the United States, the hip being the second most affected weightbearing joint. Autologous bone marrow concentrate (BMC) is a promising alternative therapy to conventional treatments, with the potential to mitigate inflammation and improve joint function.Purpose:To investigate the effectiveness of a single intra-articular BMC injection for patients with symptomatic hip OA.Study Design:Case series; Level of evidence, 4.Methods:A total of 24 patients diagnosed with symptomatic hip OA who elected to undergo a single BMC injection were prospectively enrolled in the study. Patients were excluded if they reported a preinjection Numeric Rating Scale (NRS) score for pain with activity of Results:A total of 18 hips from 16 patients (7 male and 9 female) (mean age, 57.6 ± 11; mean body mass index, 25.9 ± 3.6 kg/m2) were used in the final analysis. Significant improvements were observed in NRS pain with activity (from 8 to 4.5; P < .001) and without activity (from 5 to 1; P < .001), WOMAC (from 31 to 16; P = .006), mHHS (from 63 to 80; P = .004), and HOS-ADL (from 71 to 85; P = .014) over 6 months. At 6 months, all patients maintained their improvements and did not return to preprocedure status. BMI significantly correlated with baseline WOMAC scores ( P = .012) and inversely correlated with 6-month SF-12 Physical Component Summary ( P = .038). Tönnis grades 2 and 3 were inversely correlated with 6-week SF-12 Mental Component Summary ( P = .008) and 3-month pain with activity ( P = .032). No serious adverse events were reported from the BMC harvest or injection procedure.Conclusion:A single BMC injection can significantly improve subjective pain and function scores up to 6 months in patients with symptomatic hip OA. Further studies are warranted to evaluate BMC treatment against other therapeutics in a larger sample size and compare the biological signature profiles that may be responsible for the therapeutic effect.

Recent efforts have focused on customizing orthobiologics, such as platelet-rich plasma (PRP) and bone marrow concentrate (BMC), to improve tissue repair. We hypothesized that oral losartan (a TGF-&beta, 1 blocker with anti-fibrotic properties) could decrease TGF-&beta, 1 levels in leukocyte-poor PRP (LP-PRP) and fibrocytes in BMC. Ten rabbits were randomized into two groups (N = 5/group): osteochondral defect + microfracture (control, group 1) and osteochondral defect + microfracture + losartan (losartan, group 2). For group 2, a dose of 10mg/kg/day of losartan was administrated orally for 12 weeks post-operatively. After 12 weeks, whole blood (WB) and bone marrow aspirate (BMA) samples were collected to process LP-PRP and BMC. TGF-&beta, 1 concentrations were measured in WB and LP-PRP with multiplex immunoassay. BMC cell populations were analyzed by flow cytometry with CD31, CD44, CD45, CD34, CD146 and CD90 antibodies. There was no significant difference in TGF-&beta, 1 levels between the losartan and control group in WB or LP-PRP. In BMC, the percentage of CD31+ cells (endothelial cells) in the losartan group was significantly higher than the control group (p = 0.008), while the percentage of CD45+ cells (hematopoietic cells-fibrocytes) in the losartan group was significantly lower than the control group (p = 0.03).

To assess the utilization and efficacy of platelet-rich plasma (PRP), for the treatment of articular cartilage injury, most commonly characterized by progressive pain and loss of joint function in the setting of osteoarthritis (OA). PRP modulates the inflammatory and catabolic environment through a locally applied concentrate of platelets, leukocytes, and growth factors. Clinically, PRP has been shown to be possibly a viable treatment adjuvant for a variety of inflammatory and degenerative conditions. Recent efforts have focused on optimizing delivery methods that enable platelets to slowly degranulate their biological constituents, which may promote healing and improve OA symptoms for a longer duration. There are various factors that affect the progression of OA within joints, including inhibition of inflammatory cytokines and altering the level of enzymatic expression. PRP therapy aims to mediate inflammatory and catabolic factors in a degenerative environment through the secretion of anti-inflammatory factors and chemotaxic effects. There are a growing number of studies that have demonstrated the clinical benefit of PRP for non-operative management of OA. Additional randomized controlled trials with long-term follow-up are needed in order to validate PRP’s therapeutic efficacy in this setting. Additionally, continued basic research along with well-designed pre-clinical studies and reporting standards are necessary in order to clarify the effectiveness of PRP for cartilage repair and regeneration for future clinical applications.

Musculoskeletal injuries that disrupt the structure and function of diarthrodial joints can cause permanent biomechanical alterations and lead to a more severe, chronic condition. Despite advancements that have been made to restore tissue function and delay the need for joint replacement, there are currently no disease-modifying therapies for osteoarthritis (OA). To reduce the risk of OA, innovative preventive medicine approaches have been developed over the last decade to treat the underlying pathology. Several biological approaches are promising treatment modalities for various stages of OA owing to their minimally invasive nature and actively dynamic physiological mechanisms that attenuate tissue degradation and inflammatory responses. Individualized growth factor and cytokine therapies, tissue-engineered biomaterials, and cell-based therapies have revolutionary potential for orthopedic applications; however, the paucity of standardization and categorization of biological components and their counterparts has made it difficult to determine their clinical and biological efficacy. Cell-based therapies and tissue-engineered biologics have become lucrative in sports medicine and orthopedics; nonetheless, there is a continued effort to produce a biological treatment modality tailored to target intra-articular structures that recapitulates tissue function. Advanced development of these biological treatment modalities will potentially optimize tissue healing, regeneration, and joint preservation strategies. Therefore, the purpose of this paper is to review current concepts on several biological treatment approaches for OA.

Objectives: The possibility of preserving platelet-rich plasma (PRP) from young, healthy individuals for future use is a compelling approach to reduce or delay degenerative processes, presuming that the retention of the biological properties are maintained. The purpose of this study was to measure and compare matrix metalloproteinases (MMP) isoform concentrations between whole blood (WB), leukocyte-rich PRP (LR-PRP) inactivated (LR-I) and activated (LR-A), leukocyte-poor PRP (LP-PRP) inactivated (LP-I) and activated (LP-A). Methods: Following institutional review board approval (2017-36), 24 donors that were physically and mentally healthy were prospectively enrolled in the study. Approximately 60 mL of WB was drawn from each donor to produce inactivated and activated LP-PRP and LR-PRP using manual processing methodology, as previously described. A complete blood count for WB and inactivated PRP products was obtained to verify that concentration of platelets was achieved. WB, LP-I, and LR-I samples were set aside for immunoassay and analysis. The LP-I and LR-I products were activated with 10% calcium chloride and recombinant thrombin in a red-top 10 mL vacutainer tube. Blood fractions were either immediately assayed and analyzed (fresh) or stored at -80℃ for 24 hours, 72 hours, and 160 hours. Commercial kits (EMD Millipore) were used according to manufacturer’s instructions for protein content: MMP-1, MMP-3, MMP-9, MMP-10, and MMP-12. A standard methodology for the Luminex 200® system was used as previously published. A pairwise Wilcoxin rank test was performed for statistical calculation. Results: Twenty-two healthy donors (n = 12 females, n = 10 males) with a mean age of 37.7 (range: 21 to 60), and average BMI of 23.7 kg/m2, were used in the final analysis. MMP-1 significantly increased between fresh and 160 hours in WB (pConclusion: In this study, we evaluated the influence of short-term freezing (-80℃) on MMP concentrations in WB, inactivated PRP, and activated PRP formulations. Our results suggest that certain MMP isoforms, can either increase or decrease in response to freezing in WB, inactivated PRP, and activated PRP formulations. The development of PRP preservation approaches through minimal manipulation, without attenuating its biological properties, represents an important step in PRP mediated tissue regeneration and repair. [Figure: see text][Figure: see text][Figure: see text][Figure: see text]

Skeletal muscle is a highly interactive connective tissue that makes up a large portion of an adult's body mass. Muscles are integrated with tendons as well as other specialized structures to support physiologic and homeostatic functions. In the state of injury, muscles have the ability to innately repair themselves through various phases of tightly regulated healing that can result in fibrotic tissue development. However, during aging, these homeostatic processes are disrupted leading to sarcopenia and reduced muscle regeneration capacity. Several cell-based therapies and biologic therapies have been investigated to regenerate skeletal muscle tissue and reduce fibrosis following injury or during aging. These include platelet-rich plasma (PRP) and an acellular portion of blood known as platelet-poor plasma (PPP). However, current clinical practice recommendations for the utilization of different PRP preparations vs PPP are unclear. Recent efforts have strove to improve the understanding of the role of senescent cells and profiles in the presence of early to late stage skeletal muscle injury and fibrosis, yet targeted interventions to remove senescent cells and attenuate the secretory environment to improve muscle regeneration are still forthcoming. Therefore, the purpose of this article is to review the basic principles of skeletal muscle repair, the role of senescence in attenuated muscle regeneration, and discuss current standards and literature supporting PRP and PPP treatment for skeletal muscle repair. This review concludes with future directions to improve biologic therapies and ongoing initiatives to customize PRP and PPP preparations using Food and Drug Administration-approved medications.

Biological augmentation and therapeutics are being increasingly used in musculoskeletal and orthopaedic care. Platelet-rich plasma (PRP) is produced from centrifugation of peripheral blood, a process that concentrates platelets within autologous plasma. The process of PRP preparation is fundamental in controlling the contents, and it influences its therapeutic potential. Platelets contain alpha granules that store and release a variety of growth factors and other proteins that may augment the healing environment; PRP also has the added benefit of promoting postsurgical hemostasis. The purpose of this report was to detail our institutional preparation protocol and method of administration of PRP during hip arthroscopy.

Kaitlyn E. Whitney, Ioanna K. Bolia, Jorge Chahla, Hajime Utsunomiya, Thos A. Evans, Matthew Provencher, Peter J. MilletThe Steadman Clinic and Steadman Philippon Research Institute, Vail, CO, USAJohnny HuardThe Steadman Clinic and Steadman Philippon Research Institute, Vail, CO, USADepartment of Orthopedic Surgery, University of Texas Health Science Center at Houston, Houston, Texas, USA

Objectives: Platelet-rich plasma (PRP) is comprised of several biologically active factors that can stimulate musculoskeletal healing processes. The supernatant of PRP, known as PPP, is biologically active and may also stimulate tissue regeneration. In some instances, such as muscle injury, PPP may be preferred to PRP in order to stimulate muscle regrowth in a basic science study that was previously performed. Platelet poor plasma (PPP) is has several biologically active molecular factors that may be utilized to stimulate tissue healing. While platelet rich plasma has been previously studied and characterized, few studies have sought to quantify the biological constituents of PPP. The purpose of this study was to quantitate and assess growth factors, other chemokines, and cytokines in PPP derived from human peripheral blood that has been centrifuged. Study Design: Non-randomized, prospective cohort study; Level of evidence: 2. Methods: Peripheral blood was drawn to create PPP at three time points from sixteen healthy volunteers. Hematology analysis was conducted on the PPP to quantify the platelet fold-difference from baseline measurements. The PPP samples were immediately assayed and analyzed on the MagPix® following processing completion. Specific immunoassay kits used were human cytokine/chemokine magnetic bead panel, TGF-β magnetic bead panel, MMP magnetic bead panel 1, and MMP magnetic bead panel 2. Results: Among the biological factors tested, there was a significant positive association, defined by two factors being associated in that when one factor increases the other also increases, between BMI and the biological composition of PPP with PDGF AA, PDGF AB, MMP-1, MMP-9, MMP-13, and MMP-12 (pConclusion: PPP has several biological factors, both anabolic and catabolic, that can potentially be utilized in musculoskeletal medicine to treat various conditions, such as muscle injury. PPP is biologically active and this study characterizes its anabolic and catabolic profile. These factors are influenced by certain demographic factors such as age and body mass index (BMI). Higher BMI significantly correlated to higher levels of PDGF AA, PDGF AB, MMP-1, MMP-9, MMP-13, and MMP-12 in PPP. This supernatant of the better-studied PRP is biological active and warrants further investigation for its therapeutic potential. Platelets could change the biological composition of plasma utilized for regenerative medicine applications, but this study demonstrates that the plasma alone has biological properties that may provide benefit in treating certain musculoskeletal conditions. This study will help clinicians better understand the biological nature of PPP and may aide in the more targeted use of PPP therapeutically.

Objectives: Platelet-rich plasma (PRP) is comprised of several biologically active factors that can stimulate musculoskeletal healing processes. Non-steroidal anti-inflammatory drugs (NSAIDs) are used to treat many musculoskeletal conditions and influence platelet function. The influence of NSAIDs on the biological composition of PRP is not well known. The purpose of this study was to quantify and compare growth factors and other chemokines and cytokines in leukocyte-rich PRP (LR-PRP) between baseline, following one-week of NSAID (Naproxen) use, and after a one-week washout period. We hypothesized that Naproxen would alter both the catabolic and anabolic factors in LR-PRP. Our secondary hypothesis was that discontinuing Naproxen use for one-week would be sufficient to return LR-PRP’s molecular constituents back to baseline. Study Design: Non-randomized, prospective cohort study; Level of evidence: 2. Methods: Sixteen healthy volunteers (n=8 male and n=8 females) were enrolled in this IRB approved study. Peripheral blood was drawn at three-time points: baseline, one-week after Naproxen intake, and after a one-week washout period. Donors were instructed after the first blood-draw to ingest oral Naproxen tablets twice a day (440 mg bid) for one-week. Fifty-two anabolic and catabolic factors in LR-PRP were measured at all time points. Donor demographics were collected at the time of the initial blood-draw. To address the primary hypothesis of this study, Friedman’s test was used to identify significant differences between the baseline, Naproxen and washout time points while accounting for the repeated measures design of this study. When Friedman’s test was significant, pairwise comparisons were made using Holm’s method to control the family-wise type 1 error rate. Results: Three anabolic growth factors, platelet derived growth factor-AA (PDGF-AA, Figure 1), platelet derived growth factor-AB (PDGF-AB, Figure 1), and transforming growth factor-β1 (TGF-β1), had a significant decline from baseline (pConclusion: Naproxen use diminished several biological factors in LR-PRP; however, a one-week washout period was sufficient for the recovery of PDGF-AA, PDGF-AB, TNF-β, and IL-6 to return to baseline levels. Our results suggest that patients undergoing PRP treatment should discontinue Naproxen, and other NSAID use, at least one-week prior to harvest and injection of PRP.