Inverse agonism of retinoic acid receptors directs epiblast cells into the paraxial mesoderm lineage.

We have investigated the differentiation of paraxial mesoderm from mouse embryonic stem cells utilizing a Tbx6-EYFP/Brachyury (T)-Cherry dual reporter system. Differentiation from the mouse ESC state directly into mesoderm via Wnt pathway activation was low, but augmented by treatment with AGN193109, a pan-retinoic acid receptor inverse agonist. After five days of differentiation, T+ cells increased from 12.2% to 18.8%, Tbx6+ cells increased from 5.8% to 12.7%, and T+/Tbx6+ cells increased from 2.4% to 14.1%. The synergism of AGN193109 with Wnt3a/CHIR99021 was further substantiated by the increased expression of paraxial mesoderm gene markers Tbx6, Msgn1, Meox1, and Hoxb1. Separate to inverse agonist treatment, when mouse ESCs were indirectly differentiated into mesoderm via a transient epiblast step the efficiency of paraxial mesoderm formation markedly increased. Tbx6+ cells represented 65-75% of the total cell population after just 3 days of differentiation and the expression of paraxial mesoderm marker genes Tbx6 and Msgn increased over 100-fold and 300-fold, respectively. Further evaluation of AGN193109 treatment on the indirect differentiation protocol suggested that RARs have two distinct roles. First, AGN193109 treatment at the epiblast step and mesoderm step promoted paraxial mesoderm formation over other mesoderm and endoderm lineage types. Second, continued treatment during mesoderm formation revealed its ability to repress the maturation of presomitic mesoderm into somitic paraxial mesoderm. Thus, the continuous treatment of AGN193109 during epiblast and mesoderm differentiation steps yielded a culture where ~90% of the cells were Tbx6+. The surprisingly early effect of inverse agonist treatment at the epiblast step of differentiation led us to further examine the effect of AGN193109 treatment during an extended epiblast differentiation protocol. Interestingly, while inverse agonist treatment had no impact on the conversion of ESCs into epiblast cells based on the expression of Rex1, Fgf5, and pluripotency marker genes Oct4, Nanog, and Sox2, after three days of differentiation in the presence of AGN193109 caudal epiblast and early paraxial mesoderm marker genes, T, Cyp26a1, Fgf8, Tbx6 and Msgn were all highly up-regulated. Collectively, our studies reveal an earlier than appreciated role for RARs in epiblast cells and the modulation of their function via inverse agonist treatment can promote their differentiation into the paraxial mesoderm lineage.

Gdf5 progenitors give rise to fibrocartilage cells that mineralize via hedgehog signaling to form the zonal enthesis.

The sequence of events that leads to the formation of a functionally graded enthesis is not clearly defined. The current study demonstrates that clonal expansion of Gdf5 progenitors contributes to linear growth of the enthesis. Prior to mineralization, Col1+ cells in the enthesis appose Col2+ cells of the underlying primary cartilage. At the onset of enthesis mineralization, cells at the base of the enthesis express alkaline phosphatase, Indian hedgehog, and ColX as they mineralize. The mineralization front then extends towards the tendon midsubstance as cells above the front become encapsulated in mineralized fibrocartilage over time. The hedgehog (Hh) pathway regulates this process, as Hh-responsive Gli1+ cells within the developing enthesis mature from unmineralized to mineralized fibrochondrocytes in response to activated signaling. Hh signaling is required for mineralization, as tissue-specific deletion of its obligate transducer Smoothened in the developing tendon and enthesis cells leads to significant reductions in the apposition of mineralized fibrocartilage. Together, these findings provide a spatiotemporal map of events - from expansion of the embryonic progenitor pool to synthesis of the collagen template and finally mineralization of this template - that leads to the formation of the mature zonal enthesis. These results can inform future tendon-to-bone repair strategies to create a mechanically functional enthesis in which tendon collagen fibers are anchored to bone through mineralized fibrocartilage.

Coated Sutures.

The addition of specific proteins or growth factors onto sutures would provide a direct application of exogenous factors to promote tissue repair. The higher levels of growth factors and cytokines may optimize the healing environment and promote tissue recovery. Despite this proposed benefit, the current orthopedic literature on the use of coated sutures is limited. Although several of the published studies investigating healing improvement by coated sutures have shown promising results, these data are only based on in vitro or small animal experiments. Recent meta-analyses have reported positive effects of triclosan-coated antimicrobial sutures in regards to reduction of surgical site complications. However, biologically coated sutures are not yet widely accepted due to several unanswered questions (concentration, release kinematics, tissue reactions, etc.) in addition to the high costs of such products. Further studies are needed to demonstrate the efficacy of coated sutures in orthopedic surgery.

US definitions, current use, and FDA stance on use of platelet-rich plasma in sports medicine.

With increased utilization of platelet-rich plasma (PRP), it is important for clinicians to understand the United States, the Food and Drug Administration (FDA) regulatory role and stance on PRP. Blood products such as PRP fall under the prevue of FDA's Center for Biologics Evaluation and Research (CBER). CBER is responsible for regulating human cells, tissues, and cellular and tissue-based products. The regulatory process for these products is described in the FDA's 21 CFR 1271 of the Code of Regulations. Under these regulations, certain products including blood products such as PRP are exempt and therefore do not follow the FDA's traditional regulatory pathway that includes animal studies and clinical trials. The 510(k) application is the pathway used to bring PRP preparation systems to the market. The 510(k) application allows devices that are "substantially equivalent" to a currently marketed device to come to the market. There are numerous PRP preparation systems on the market today with FDA clearance; however, nearly all of these systems have 510(k) clearance for producing platelet-rich preparations intended to be used to mix with bone graft materials to enhance bone graft handling properties in orthopedic practices. The use of PRP outside this setting, for example, an office injection, would be considered "off label." Clinicians are free to use a product off-label as long as certain responsibilities are met. Per CBER, when the intent is the practice of medicine, clinicians "have the responsibility to be well informed about the product, to base its use on firm scientific rationale and on sound medical evidence, and to maintain records of the product's use and effects." Finally, despite PRP being exempted, the language in 21 CFR 1271 has caused some recent concern over activated PRP; however to date, the FDA has not attempted to regulate activated PRP. Clinicians using activated PRP should be mindful of these concerns and continued to stay informed.

The enthesis: a review of the tendon-to-bone insertion.

The integration of tendon into bone occurs at a specialized interface known as the enthesis. The fibrous tendon to bone enthesis is established through a structurally continuous gradient from uncalcified tendon to calcified bone. The enthesis exhibits gradients in tissue organization classified into four distinct zones with varying cellular compositions, mechanical properties, and functions in order to facilitate joint movement. Damage to tendinous insertions is common in the field of orthopaedic medicine and often involves surgical intervention that requires the attempted recreation of the natural organization of tendon into bone. The difficulty associated with recreating the distinct organization may account for the surgical challenges associated with reconstruction of damaged insertion sites. These procedures are often associated with high failure rates and consequently require revision procedures. Management of tendinous injuries and reconstruction of the insertion site is becoming a popular topic in the field of orthopaedic medicine.

Growth factor delivery vehicles for tendon injuries: Mesenchymal stem cells and Platelet Rich Plasma.

BACKGROUND: tendon tissue shows limited regeneration potential with formation of scar tissue and inferior mechanical properties. The capacity of several growth factors to improve the healing response and decrease scar formation is described in different preclinical studies. Besides the application of isolated growth factors, current research focuses on two further strategies to improve the healing response in tendon injuries: platelet rich plasma (PRP) and mesenchymal stem cells (MSCs). OBJECTIVE: the present review focuses on these two options and describes their potential to improve tendon healing. RESULTS: in vitro experiments and animal studies showed promising results for the use of PRP, however clinical controlled studies have shown a tendency of reduced pain related symptoms but no significant differences in overall clinical scores. On the other hand MSCs are not totally arrived in clinical use so that there is still a lack of randomized controlled trials. In basic research experiments they show an extraordinary paracrine activity, anti-inflammatory effect and the possibility to differentiate in tenocytes when different activating-factors are added. CONCLUSION: preclinical studies have shown promising results in improving tendon remodeling but the comparability of current literature is difficult due to different compositions. PRP and MSCs can act as efficient growth factor vehicles, however further studies should be performed in order to adequate investigate their clinical benefits in different tendon pathologies.

Rotational and translational stability of different methods for direct acromioclavicular ligament repair in anatomic acromioclavicular joint reconstruction.

BACKGROUND: Many reconstructions of acromioclavicular (AC) joint dislocations have focused on the coracoclavicular (CC) ligaments and neglected the functional contribution of the AC ligaments and the deltotrapezial fascia. PURPOSE: To compare the modifications of previously published methods for direct AC reconstruction in addition to a CC reconstruction. The hypothesis was that there would be significant differences within the variations of surgical reconstructions. STUDY DESIGN: Controlled laboratory study. METHODS: A total of 24 cadaveric shoulders were tested with a servohydraulic testing system. Two digitizing cameras evaluated the 3-dimensional movement. All reconstructions were based on a CC reconstruction using 2 clavicle tunnels and a tendon graft. The following techniques were used to reconstruct the AC ligaments: a graft was shuttled underneath the AC joint back from anterior and again sutured to the acromial side of the joint (group 1), a graft was fixed intramedullary in the acromion and distal clavicle (group 2), a graft was passed over the acromion and into an acromial tunnel (group 3), and a FiberTape was fixed in a cruciate configuration (group 4). Anterior, posterior, and superior translation, as well as anterior and posterior rotation, were tested. RESULTS: Group 1 showed significantly less posterior translation compared with the 3 other groups (P < .05) but did not show significant differences compared with the native joint. Groups 3 and 4 demonstrated significantly more posterior translation than the native joint. Group 1 showed significantly less anterior translation compared with groups 2 and 3. Group 3 demonstrated significantly more anterior translation than the native joint. Group 1 demonstrated significantly less superior translation compared with the other groups and with the native joint. The AC joint of group 1 was pulled apart less compared with all other reconstructions. Only group 1 reproduced the native joint for the anterior rotation at the posterior marker. Group 4 showed significantly increased distances for all 3 measure points when the clavicle was rotated posteriorly. CONCLUSION: Reconstruction of the AC ligament by direct wrapping and suturing of the remaining graft around the AC joint (group 1) was the most stable method and was the only one to show anterior rotation comparable with the native joint. In contrast, the transacromial technique (group 3) showed the most translation and rotation. CLINICAL RELEVANCE: An anatomic repair should address both the CC ligaments and the AC ligaments to control the optimal physiologic function (translation and rotation).

Learning about PRP using cell-based models.

Studies using in vitro cell models enable evaluation of the effects of different PRP products under very controlled and standardized conditions. Therefore the results of such studies build the basis for understanding the variable results of clinical studies on the use of PRPs. The main lessons learned through the use of in vitro cell models are that many different PRP products exist and researchers have to report on component variation within each product. These different products may have distinctive effects on the various cells treated in musculoskeletal injuries; therefore, some products might be more beneficial in certain indication than others. In its utilization in cell models, PRP may generate a variety of positive effects on cell proliferation, recovery, and inflammatory response. There might also be a benefit to adding PRP to current pharmacological therapies (e.g. corticosteroids) to prevent their commonly known negative effects on e.g. tendon and cartilage tissue.

Properties of biologic scaffolds and their response to mesenchymal stem cells.

PURPOSE: The purpose of this study was to examine, in vitro, the cellular response of human mesenchymal stem cells (MSCs) to sample types of commercially available scaffolds in comparison with control, native tendon tissue (fresh-frozen rotator cuff tendon allograft). METHODS: MSCs were defined by (1) colony-forming potential; (2) ability to differentiate into tendon, cartilage, bone, and fat tissue; and (3) fluorescence-activated cell sorting analysis (CD73, CD90, CD45). Samples were taken from fresh-frozen human rotator cuff tendon (allograft), human highly cross-linked collagen membrane (Arthroflex; LifeNet Health, Virginia Beach, VA), porcine non-cross-linked collagen membrane (Mucograft; Geistlich Pharma, Lucerne, Switzerland), a human platelet-rich fibrin matrix (PRF-M), and a fibrin matrix based on platelet-rich plasma (ViscoGel; Arthrex, Naples, FL). Cells were counted for adhesion (24 hours), thymidine assay for cell proliferation (96 hours), and live/dead stain for viability (168 hours). Histologic analysis was performed after 21 days, and the unloaded scaffolds were scanned with electron microscopy. RESULTS: MSCs were successfully differentiated into all cell lines. A significantly greater number of cells adhered to both the non-cross-linked porcine collagen scaffold and PRF-M. Cell activity (proliferation) was significantly higher in the non-cross-linked porcine collagen scaffold compared with PRF-M and fibrin matrix based on platelet-rich plasma. There were no significant differences found in the results of the live/dead assay. CONCLUSIONS: Significant differences in the response of human MSCs to biologic scaffolds existed. MSC adhesion, proliferation, and scaffold morphology evaluated by histologic analysis and electron microscopy varied throughout the evaluated types of scaffolds. Non-cross-linked porcine collagen scaffolds showed superior results for cell adhesion and proliferation, as well as on histologic evaluation. CLINICAL RELEVANCE: This study enables the clinician and scientist to choose scaffold materials according to their specific interaction with MSCs.

Variability of platelet-rich plasma preparations.

Platelet-rich plasma (PRP) as a clinical treatment for bone, muscle, tendon, and cartilage injury has gained popularity in the field of orthopedic sports medicine. The use of a patient's own blood is an appealing aspect of PRP treatment, as the resulting plasma preparation is considered relatively benign in comparison with more common, potentially caustic treatments such as corticosteroids and anesthetics. Although appealing, the autologous nature of PRP introduces variability to plasma preparations, creating challenges for both the researcher and the clinician. Differences in patients at the time of blood draw result in plasma preparations that vary within as well as between patients. This variability is compounded by the multitude of protocols and devices available for procuring PRP. The variability of components and its effects on dosage should be considered in single or consecutive treatments of PRP.

The future role of mesenchymal stem cells in the management of shoulder disorders.

PURPOSE: Biologics may help to optimize the healing environment after rotator cuff repair. Mesenchymal stem cells (MSCs) may have the potential to regenerate a physiological enthesis, thereby improving healing at the repair site after rotator cuff repair. METHODS: The PubMed database was searched in May 2013. Only in vivo and in vitro studies reporting on stem cell use in the rotator cuff of humans or animals were included. Exclusion criteria consisted of the following: Level V evidence, systematic reviews, and studies reporting preliminary results. RESULTS: This query resulted in 141 citations. Of these, 90 were excluded based on the title of the study. A final group of 17 studies was included in this review (9 in vivo animal studies, 5 in vitro human studies, 1 in vitro animal study, 1 study reporting in vitro human and in vivo animal results, and 1 study reporting on clinical outcomes of human patients). CONCLUSIONS: The current literature regarding therapeutic use of MSCs in shoulder surgery is limited. Although in vivo animal studies have shown some promising approaches to enhance tendon-to-bone healing, the use of MSCs for shoulder surgery should still be regarded as an experimental technique. Further basic and clinical research is needed until a procedure can be defined for the routine use of these cells in shoulder surgery.

The effect of ketorolac tromethamine, methylprednisolone, and platelet-rich plasma on human chondrocyte and tenocyte viability.

PURPOSE: The purpose of this study was to evaluate the effect on cell viability of the isolated and combined use of allogeneic platelet-rich plasma (PRP) and ketorolac tromethamine on human chondrocytes and tenocytes in a highly controlled in vitro environment. METHODS: PRP was produced from 8 subjects. Human chondrocytes (Lonza, Hopkinton, MA) and tenocytes isolated from samples of the long head of the biceps tendons were treated in culture with PRP, ketorolac tromethamine, and methylprednisolone, both alone and in combination. Control samples were treated in media containing 2% or 10% fetal bovine serum (FBS). Cells were exposed for 1 hour. Luminescence assays were obtained to examine cell viability after 24 hours and long-term effects on cell viability after 120 hours. Radioactive thymidine assay was used to measure proliferation after 120 hours. RESULTS: For chondrocytes, cell viability (120 hours) increased significantly with the treatment of PRP alone (43,949 ± 28,104 cells; P < .001) and with the combination of ketorolac tromethamine and PRP (43,276 ± 31,208; P < .001), compared with the 2% FBS group (7,397 ± 470). Cell viability decreased significantly after exposure to methylprednisolone (1,323 ± 776; P < .001) and its combination with PRP (4,381 ± 5,116; p < .001). For tenocytes, cell viability (120 hours) was significantly higher with the treatment of PRP (61,287 ± 23,273; P < .001) and the combined treatment of ketorolac tromethamine and PRP (52,025 ± 17,307; P < .001), compared with the 2% FBS group (23,042 ± 2,973). Cell viability decreased significantly after exposure to methylprednisolone (3,934 ± 1,791; P = .001) and its combination with PRP (5,201 ± 2,834; P = .003), compared with 2% FBS. CONCLUSIONS: Tendon and cartilage cells showed increased cell viability after an exposure to allogeneic PRP and ketorolac tromethamine. Exposure to methylprednisolone alone decreased cell viability, and addition of PRP could partially reverse this negative effect. CLINICAL RELEVANCE: Intra-articular injections of pain-modifying or anti-inflammatory drugs are routinely given in orthopaedic practice. Among the many agents available for intra-articular injection, corticosteroids and local anesthetics are the most common in clinical practice. Potential detrimental side effects of intra-articular injections of corticosteroids and local anesthetics have prompted investigation into alternative treatment options such as combinations of PRP and ketorolac tromethamine. In vitro evaluation of their effect on cell viability might build a basis for further translational research and clinical application.

Comparison of mesenchymal stem cells (osteoprogenitors) harvested from proximal humerus and distal femur during arthroscopic surgery.

PURPOSE: The aim of this study was to examine the relations between age, gender, and number of viable mesenchymal stem cells (MSCs) in concentrated bone marrow (BM) obtained from the proximal humerus and distal femur during arthroscopic surgery. METHODS: BM was aspirated from either the proximal humerus (n = 55) or distal femur (n = 29) during arthroscopic surgery in 84 patients (51.3 ± 11.6 years). MSCs were obtained from fractionated bone marrow after a 5-minute spin at 1,500 rpm. Volume of BM and number of nucleated cells (NCs) were calculated, and samples were cultured for 6 days, after which point colony-forming units (CFUs) were quantified and fluorescence-activated cell sorting (FACS) analysis was performed. Simple linear regression was used to explore relations between age, gender, volume of aspirated BM, and MSCs per milliliter. RESULTS: BM aspirations yielded a mean quantity of 22.6 ± 12.3 mL. After centrifugation, 30.0 ± 16.7 × 10(6) nucleated cells/mL of concentrated BM were harvested. The proximal humerus provided 38.7 ± 52.6 × 10(6), and the distal femur, 25.9 ± 14.3 × 10(6), for an overall 766.3 ± 545.3 MSCs/mL of concentrated BM (proximal humerus: 883.9 ± 577.6, distal femur: 551.3 ± 408.1). Values did not significantly differ by age, gender, or donor site. CONCLUSIONS: Arthroscopic aspiration of bone marrow from the proximal humerus and distal femur is a reproducible technique and yields reliable concentrations of MSCs. The use of an intraoperative concentration method resulted in consistent amounts of MSCs in all clinically relevant age groups without a significant drop of the number of isolated MSCs. CLINICAL RELEVANCE: Human MSCs derived from concentrated bone marrow aspirate are a promising biological addition that may have practical use in the future of soft tissue augmentation. Arthroscopic techniques for bone marrow aspiration that do not require an additional surgical site for aspiration (e.g., iliac crest) or a second operative procedure may facilitate future use of MSCs in arthroscopic surgery.

Platelet-rich plasma: the PAW classification system.

Platelet-rich plasma (PRP) has been the subject of hundreds of publications in recent years. Reports of its effects in tissue, both positive and negative, have generated great interest in the orthopaedic community. Protocols for PRP preparation vary widely between authors and are often not well documented in the literature, making results difficult to compare or replicate. A classification system is needed to more accurately compare protocols and results and effectively group studies together for meta-analysis. Although some classification systems have been proposed, no single system takes into account the multitude of variables that determine the efficacy of PRP. In this article we propose a simple method for organizing and comparing results in the literature. The PAW classification system is based on 3 components: (1) the absolute number of Platelets, (2) the manner in which platelet Activation occurs, and (3) the presence or absence of White cells. By analyzing these 3 variables, we are able to accurately compare publications.

Stability of double-row rotator cuff repair is not adversely affected by scaffold interposition between tendon and bone.

BACKGROUND: Rotator cuff reconstructions may be improved by adding growth factors, cells, or other biologic factors into the repair zone. This usually requires a biological carrier (scaffold) to be integrated into the construct and placed in the area of tendon-to-bone healing. This needs to be done without affecting the constructs mechanics. Hypothesis/ PURPOSE: The hypothesis was that scaffold placement, as an interposition, has no adverse effects on biomechanical properties of double-row rotator cuff repair. The purpose of this study was to examine the effect of scaffold interposition on the initial strength of rotator cuff repairs. STUDY DESIGN: Controlled laboratory study. METHODS: Twenty-five fresh-frozen shoulders (mean age: 65.5 ± 8.9 years) were randomly assigned to 5 groups. Groups were chosen to represent a broad spectrum of commonly used scaffold types: (1) double-row repair without augmentation, (2) double-row repair with interposition of a fibrin clot (Viscogel), (3) double-row repair with interposition of a collagen scaffold (Mucograft) between tendon and bone, (4) double-row repair with interposition of human dermis patch (ArthroFlex) between tendon and bone, and (5) double-row repair with human dermis patch (ArthroFlex) placed on top of the repair. Cyclic loading to measure displacement was performed to 3000 cycles at 1 Hz with an applied 10- to 100-N load. The ultimate load to failure was determined at a rate of 31 mm/min. RESULTS: There were no significant differences in mean displacement under cyclic loading, slope, or energy absorbed to failure between all groups (P = .128, P = .981, P = .105). Ultimate load to failure of repairs that used the collagen patch as an interposition (573.3 ± 75.6 N) and a dermis patch on top of the reconstruction (575.8 ± 22.6 N) was higher compared with the repair without a scaffold (348.9 ± 98.8 N; P = .018 and P = .025). No significant differences were found for repairs with the fibrin clot as an interposition (426.9 ± 103.6 N) and the decellularized dermis patch as an interposition (469.9 ± 148.6 N; P = .73 and P = .35). CONCLUSION: Scaffold augmentation did not adversely affect the zero time strength of the tested standard double-row rotator cuff repairs. An increased ultimate load to failure was observed for 2 of the augmentation methods (collagen patch as an interposition and decellularized dermis patch on top of the reconstruction) compared with the nonaugmented repairs. CLINICAL RELEVANCE: Scaffolds intended for application of growth factors or cellular components in a repair situation did not adversely jeopardize the stability of the operative construct.