Connective Tissue Progenitor Cells Research Articles

Orthobiologics: Current Status in 2023 and Future Outlook.

Orthobiologic agents, including platelet-rich plasma, connective tissue progenitor cells derived from bone marrow, adipose, and other tissues, and purified cytokines and small peptides, have tremendous potential to target deficiencies in soft-tissue healing. The principal limitation currently is the variability in the composition and biologic activity of orthobiologic formulations, making it difficult to choose the optimal treatment for a specific tissue or pathology. Current data suggest that orthobiologics are "symptom-modifying," but there is little evidence that they can lead to true tissue regeneration ("structure-modifying"). A critically important need at this time is to identify sentinel markers of potency and biologic activity for different orthobiologic formulations so that we can match the treatment to the desired biologic effect for a specific tissue or pathology. Improved understanding of the underlying cellular and molecular mechanisms of tissue degeneration and repair will allow a precision medicine approach where we can choose the optimal orthobiologic treatment of specific orthopaedic problems. It is important for the clinician to be aware of the evolving regulatory status of orthobiologic treatments. Emerging therapies such as the use of exosomes and gene therapy approaches hold great promise as improved methods to both treat symptoms and affect tissue regeneration.

Harvest and Application of Bone Marrow Aspirate Concentrate to Address Acetabular Chondral Damage During Hip Arthroscopy.

Harvest and Application of Bone Marrow Aspirate Concentrate to Address Acetabular Chondral Damage During Hip Arthroscopy.

Subacromial bursa increases the failure force in a mouse model of supraspinatus detachment and repair.

It has been shown that subacromial bursa (SAB) harbors connective tissue progenitor cells. The purpose of this study was to evaluate the effects of implantation of SAB-derived cells (SBCs) suspended in a fibrin sealant bead and implantation of SAB tissue at rotator cuff repair site on biomechanical properties of the repair in a mouse (C57Bl/6) model of supraspinatus tendon (ST) detachment and repair. Part 1: Murine SAB tissue was harvested and cultured. Viability of SBCs suspended in 10 μL of fibrin sealant beads was confirmed invitro and invivo. Eighty mice underwent right ST detachment and repair augmented with either fibrin sealant bead (control group) or fibrin sealant bead with 100,000 SBCs (study group) applied at the repair site. Part 2: 120 mice underwent right ST detachment and repair and were randomized equally into 4 groups: (1) a tissue group, which received a piece of freshly harvested SAB tissue; (2) a cell group, which received SBCs suspended in fibrin sealant bead; (3) a fibrin sealant group, which received plain fibrin sealant bead without cells; and (4) a control group, which received nothing at the ST repair site. An equal number of mice in each group were killed at 2 and 4 weeks. Specimens underwent biomechanical testing to evaluate failure force (part 1 and 2) and histologic analysis of the repair site (part 1 only). Part 1: The mean failure force in the study group was significantly higher than controls at 2 and 4 weeks (3.25±1.03 N vs. 2.43±0.56 N, P=.01, and 4.08±0.99 N vs. 3.02±0.8 N, P=.004, respectively). Mean cell density of the ST at the repair site was significantly lower in the study group at 2 weeks than in controls (18,292.13±1706.41 vs. 29,501.90±3627.49, P=.001). Study group specimens had lower proteoglycan contents than controls, but this difference was not statistically significant. Part 2: There was no difference in failure force between cell and tissue groups at the 2- and 4-week time points (P=.994 and P=.603, respectively). There was no difference in failure force between fibrin sealant bead and control groups at the 2- and 4-week time points (P=.978 and P=.752, respectively). This study shows that the application of SBCs and SAB tissue at the rotator cuff repair site increases the strength of repair in a murine model of rotator cuff detachment and repair.

Percutaneous autologous bone marrow concentrate for knee osteoarthritis: patient-reported outcomes and progenitor cell content

PurposeKnee osteoarthritis (OA) is a common, progressively debilitating joint disease, and the intra-articular injection of autologous bone marrow concentrate (BMC) may offer a minimally invasive method of harnessing the body’s own connective tissue progenitor cells to counteract accompanying degenerative effects of the disease. However, the extent to which the progenitor cell content of BMC influences treatment outcomes is unclear. We sought to determine whether patient-reported outcome measures associated with BMC treatment for knee OA are related to the concentration of progenitor cells provided.MethodsIn the present study, 65 patients (72 knees) underwent treatment for knee OA with autologous BMC and self-reported their outcomes for up to one year using follow-up questionnaires tracking function, pain, and percent improvement. A small fraction of each patient’s BMC sample was reserved for quantification with a haematological analyzer and cryopreserved for subsequent analysis of potential connective tissue progenitor cells using a colony-forming unit fibroblast (CFU-F) assay.ResultsPatients reported significant increases in function and overall percent improvement in addition to decreases in pain relative to baseline levels following treatment with autologous BMC that persisted through 12 months. Patients reporting improved outcomes (46 of 72 knees) received BMC injections having higher CFU-F concentrations than non-responding patients (21.1×103 ± 12.4×103 vs 14.3×103 ± 7.0 x103 CFU-F per mL). A progenitor cell concentration of 18×103 CFU-F per mL of BMC was found to best differentiate responders from non-responders.ConclusionThis study provides supportive evidence for using autologous BMC in the minimally invasive treatment of knee OA and suggests that increased progenitor cell content leads to improved treatment outcomes.Trial registrationClinicalTrials.gov Identifier: NCT03011398, 1/7/17

Investigating the Presence of Connective Tissue Progenitor Cells within the Retrocalcaneal Bursa

Category:Ankle; Basic Sciences/Biologics; OtherIntroduction/Purpose:The purpose of this study was to evaluate the presence of connective tissue progenitor cells within the retrocalcaneal bursa. The study evaluation included an assessment of the viability of connective tissue progenitor cells, their proliferative potential, and their ability to differentiate into osteoblasts, adipocytes, and chondrocytes.Methods:The retrocalcaneal bursa samples were excised from 10 patients (age: 51.5 +- 10.8 years) undergoing Achilles surgery in which the retrocalcaneal bursa is routinely excised. Bursal tissue was processed by digesting cells with collagenase and obtaining nucleated cell counts and cellular concentrations. The cells were cultured, and differentiated into osteoblasts, adipocytes, and chondrocytes. Analysis of bursal derived cells consisted of fluorescent activated cell sorting (FACS) analysis, cellular proliferation and viability assay, and analysis of differentiation into osteoblasts, adipocytes, and chondrocytes. The CTP differentiation was confirmed using histology staining to qualitatively express differentiation, and qPCR to quantify gene expression.Results:Cell migration at 3 weeks on average was found to be 3.84x107 nucleated cells/gram of tissue and nucleated cellular concentration on average was found to be 6.28x105 cells/mL of suspension. The proliferation data showed high levels of proliferation on average of 1.72 +-0.58. The FACS analysis showed a high percentage of positive surface markers for CTPs measuring greater than 96% (CD105, CD90, CD73) and measuring < 1.1% for negative surface markers (CD45 and CD31). Differentiation into osteoblasts, adipocytes, and chondrocytes were stained appropriately displaying differentiation.Conclusion:The retrocalcaneal bursa is a novel source of connective tissue progenitor cells. This is the first study to our knowledge analyzing the retrocalcaneal bursa as a novel source for CTPs, making it a potential augment to expedite the healing process of the Achilles tendon. Overall, it is unclear what the role of the CTPs within the bursa is, however, they may help to reduce the extensive healing time for the Achilles tendon thereby returning patients with Achilles tendon pathology back to functional status more promptly.

Efficacy of Arthroscopic Shavers for the Retrieval and Processing of Connective Tissue Progenitor Cells from Subacromial Bursal Tissue.

The purpose of this study is to determine if arthroscopic shavers can effectively collect and process connective tissue progenitor (CTP) cells from subacromial bursal tissue for utilization in rotator cuff repair augmentation. Subacromial bursal tissue was collected and processed using two arthroscopic shavers, Shaver A and Shaver B, in 10 patients undergoing arthroscopic rotator cuff repair. Each shaver was used in a random order for the same patient. Tissue samples underwent testing for cellular proliferation, cellular concentration, number of colony-forming units (CFU), live/dead assay, fluorescence-activated cells sorting (FACS) analysis, cytokine analysis, and growth factor analysis. Shaver A produced more CFUs compared to Shaver B (210.3 vs. 125.9; p < 0.001). At 3 weeks, cells collected via Shaver A had greater cellular proliferation (0.35 vs. 0.51; p < 0.001) as well as more viable cells (214,773 vs. 132,356 cells/gram; p < 0.001). Tissue collected with Shaver B had greater amounts of the cytokines MMP-1 (3741 vs. 5500 pg/mL; p < 0.001), MMP-3 (1131 vs. 1871 pg/mL; p < 0.001), and MMP-13 (179 vs. 401 pg/mL; p < 0.001), while those collected with Shaver A had greater vascular endothelial growth factor (VEGF) (47.8 vs. 9.0 pg/mL; p < 0.05). Arthroscopic shavers are capable of harvesting and processing CTP cells from subacromial bursal tissue. Different shavers may produce different yields of viable CTP cells.

In Vitro Cellular Strain Models of Tendon Biology and Tenogenic Differentiation.

Research has shown that the surrounding biomechanical environment plays a significant role in the development, differentiation, repair, and degradation of tendon, but the interactions between tendon cells and the forces they experience are complex. In vitro mechanical stimulation models attempt to understand the effects of mechanical load on tendon and connective tissue progenitor cells. This article reviews multiple mechanical stimulation models used to study tendon mechanobiology and provides an overview of the current progress in modelling the complex native biomechanical environment of tendon. Though great strides have been made in advancing the understanding of the role of mechanical stimulation in tendon development, damage, and repair, there exists no ideal in vitro model. Further comparative studies and careful consideration of loading parameters, cell populations, and biochemical additives may further offer new insight into an ideal model for the support of tendon regeneration studies.

Trochanteric Bursa Is a Source of Connective Tissue Progenitor Cells

PurposeTo investigate the presence of connective tissue progenitor cells (CTPs) in the trochanteric bursa harvested over the gluteus medius muscle belly and tendon during open hip procedures.MethodsTrochanteric bursa samples from nine patients (63.1 ± 8.6 years) undergoing total hip arthroplasty for primary osteoarthritis were obtained from 2 sites: over the gluteus medius tendon at the greater trochanter and over the muscle belly. Bursal tissue was digested with collagenase and grown in culture. The nucleated cell count, cellular concentration, cellular proliferation, fluorescence-activated cell sorting (FACS) analysis, and differentiation using immunostaining and quantitative polymerase chain reaction (PCR) were used to determine and quantify the presence of CTPs.ResultsBursa-derived CTPs were identified in all harvested samples. At t = 0, there was no difference in nucleated cell count over muscle and tendon (1.69 ± 1.26 × 108 and 1.41 ± 1.12 × 108 cells/g, respectively; P = .162). Similarly, the cellular concentration at 3 weeks was not significantly different between bursa harvested over muscle and tendon (6.61 ± 5.14 × 106 and 5.58 ± 4.70 × 106 cells/g, respectively; P = .532). High cellular proliferation was identified for both bursal tissue overlying muscle and tendon (2.28 ± .95 and 1.66 ± 1.05, respectively; P = .194). FACS analysis revealed high positivity rates (>95%) of CTP-specific surface epitopes (CD105, CD90, and CD73) and low positivity rates (<1.3%) of negative markers (CD45, CD31). Osteogenic, adipogenic, and chondrogenic differentiation potential was demonstrated with immunostaining and quantitative PCR for gene expression.ConclusionsConnective tissue progenitor cells are found in the trochanteric bursa overlying the muscle and tendon of the hip abductors.Clinical RelevanceDuring open hip procedures, the trochanteric bursa is often partially excised to identify muscular boundaries and tissue planes for surgical exposure. The function of the trochanteric bursa remains largely unknown. However, this tissue is a source of connective tissue progenitor cells, which may be important in the healing response of surgically repaired abductor tendons.

Analysis of Patient Factors Affecting In Vitro Characteristics of Subacromial Bursal Connective Tissue Progenitor Cells during Rotator Cuff Repair.

Unsatisfactory failure rates following rotator cuff (RC) repair have led orthopaedic surgeons to explore biological augmentation of the healing enthesis. The subacromial bursa (SB) contains abundant connective tissue progenitor cells (CTPs) that may aid in this process. The purpose of the study was to investigate the influence of patient demographics and tear characteristics on the number of colony-forming units (CFUs) and nucleated cell count (NCC) of SB-derived CTPs. In this study, we harvested SB tissue over the supraspinatus tendon and muscle in 19 patients during arthroscopic RC repair. NCC of each sample was analyzed on the day of the procedure. After 14 days, CFUs were evaluated under a microscope. Spearman’s rank correlation coefficient was then used to determine the relationship between CFUs or NCC and patient demographics or tear characteristics. The study found no significant correlation between patient demographics and the number of CFUs or NCC of CTPs derived from the SB (p > 0.05). The study did significantly observe that increased tear size was negatively correlated with the number of CFUs (p < 0.05). These results indicated that increased tear size, but not patient demographics, may influence the viability of CTPs and should be considered when augmenting RCrepairs with SB.

Colony Forming Potential and Protein Composition of Commercial Umbilical Cord Allograft Products in Comparison With Autologous Orthobiologics

Background: Umbilical cord (UC) connective tissues contain plastic-adherent, colony forming unit–fibroblasts (CFU-Fs) amenable to culture expansion for potential therapeutic use. Recently, UC-derived allograft products have been made available to practitioners in orthopaedics and other specialties, by companies purporting “stem cell”–based healing. However, such marketing claims conflict with existing regulations for these human tissues, generating questions over the cellular and protein composition of current commercially available UC allograft products. Purpose: To evaluate commercial UC allograft products for viable cells, CFU-Fs, and protein makeup. Study Design: Descriptive laboratory study. Methods: Five commercial UC allograft products claiming to contain viable, undescribed “stem cells,” 2 obtained from UC blood (UCB) and 3 from UC tissue (UCT), were analyzed. Image-based methods were used to measure cell concentration and viability, a traditional CFU-F assay was used to evaluate in vitro behavior indicative of a connective tissue progenitor cell phenotype often referred to as mesenchymal stem/stromal cells, and quantitative immunoassay arrays were used to measure a combination of cytokines and growth factors. Bone marrow concentrate (BMC) and plasma derived from the blood and bone marrow of middle-aged individuals served as comparative controls for cell culture and protein analyses, respectively. Results: Viable cells were identified within all 5 UC allograft products, with those derived from UCB having greater percentages of living cells (40%-59%) than those from UCT (1%-22%). Compared with autologous BMC (>95% viability and >300 million living cells), no CFU-Fs were observed within any UC allograft product (<15 million living cells). Moreover, a substantial number of proteins, particularly those within UCB allograft products, were undetectable or present at lower concentrations compared with blood and bone marrow plasma controls. Interestingly, several important growth factors and cytokines, including basic fibroblast growth factor, hepatocyte growth factor, interleukin-1 receptor antagonist, and osteoprotegerin, were most prevalent in 1 or more UCT allograft products as compared with blood and bone marrow plasma. Conclusion: CFU-Fs, often referred to as stem cells, were not found within any of the commercial UC allograft products analyzed, and clinicians should remain wary of marketing claims stating otherwise. Clinical Relevance: Any therapeutic benefit of current UC allograft products in orthopaedic medicine is more likely to be attributed to their protein composition (UCT > UCB) or inclusion of cells without colony forming potential (UCB > UCT).

Nonoperative and Operative Soft-Tissue and Cartilage Regeneration and Orthopaedic Biologics of the Knee: An Orthoregeneration Network (ON) Foundation Review

Orthoregeneration is defined as a solution for orthopedic conditions that harnesses the benefits of biology to improve healing, reduce pain, improve function, and optimally, provide an environment for tissue regeneration. Options include: drugs, surgical intervention, scaffolds, biologics as a product of cells, and physical and electro-magnetic stimuli. The goal of regenerative medicine is to enhance the healing of tissue after musculoskeletal injuries as both isolated treatment and adjunct to surgical management, using novel therapies to improve recovery and outcomes. Various orthopaedic biologics (orthobiologics) have been investigated for the treatment of pathology involving the knee, including symptomatic osteoarthritis and chondral injuries, as well as injuries to tendon, meniscus, and ligament, including the anterior cruciate ligament. Promising and established treatment modalities include hyaluronic acid (HA) in liquid or scaffold form; platelet-rich plasma (PRP); bone marrow aspirate (BMA) comprising mesenchymal stromal cells (MSCs), hematopoietic stem cells, endothelial progenitor cells, and growth factors; connective tissue progenitor cells (CTPs) including adipose-derived mesenchymal stem cells (AD-MSCs) and tendon-derived stem cells (TDSCs); matrix cell–based therapy including autologous chondrocytes or allograft; vitamin D; and fibrin clot. Future investigations should standardize solution preparations, because inconsistent results reported may be due to heterogeneity of HA, PRP, BMAC, or MSC preparations and regimens, which may inhibit meaningful comparison between studies to determine the true efficacy and safety for each treatment.

Spatial patterns of heterotopic ossification in fibrodysplasia ossificans progressiva correlate with anatomic temperature gradients.

Progressive heterotopic ossification (HO) is a hallmark of fibrodysplasia ossificans progressiva (FOP); however, this tissue transformation is not random. Rather, we noticed that HO in FOP progresses in well-defined but inexplicable spatial and temporal patterns that correlate precisely with infrared thermographs of the human body. FOP is caused by gain-of-function mutations in Activin A receptor type I (ACVR1/ALK2), a bone morphogenetic protein (BMP) type I receptor kinase. As with all enzymes, the activity of ACVR1 is temperature-dependent. We hypothesized that connective tissue progenitor cells that express the common heterozygous ACVR1R206H mutation (FOP CTPCs) exhibit a dysregulated temperature response compared to control CTPCs and that the temperature of FOP CTPCs that initiate and sustain HO at various anatomic sites determines, in part, the anatomic distribution of HO in FOP. We compared BMP pathway signaling at a range of physiologic temperatures in primary CTPCs isolated from FOP patients (n=3) and unaffected controls (n=3) and found that BMP pathway signaling and resultant chondrogenesis were amplified in FOP CTPCs compared to control CTPCs (p<0.05). We conclude that the anatomic distribution of HO in FOP may be due, in part, to a dyregulated temperature response in FOP CTPCs that reflect anatomic location. While the association of temperature gradients with spatial patterns of HO in FOP does not demonstrate causality, our findings provide a paradigm for the physiologic basis of the anatomic distribution of HO in FOP and unveil a novel therapeutic target that might be exploited for this disabling condition.

Nucleated Cell Count Has Negligible Predictive Value for the Number of Colony-Forming Units for Connective Tissue Progenitor Cells (Stem Cells) in Bone Marrow Aspirate Harvested From the Proximal Humerus During Arthroscopic Rotator Cuff Repair

To evaluate whether nucleated cell count (NCC) could serve as an approximation for the number of colony-forming units (CFUs) in concentrated bone marrow aspirate (cBMA) obtained from the proximal humerus. Bone marrow aspirate (BMA) was harvested from the proximal humerus in 96 patients (mean age 56.2 ± 7.0 years) during arthroscopic rotator cuff repair. Following concentration of the aspirate, nucleated cells of each sample were counted. The total number of CFUs was evaluated under the microscope at their first appearance, usually after 5 to 10 days in culture. Fluorescence-activated cell sorting analysis and assays for osteogenic, adipogenic, and chondrogenic differentiation were performed. Linear regression was assessed to predict the number of CFUs by using NCC. Age, sex, and body mass index (BMI) were evaluated as independent variables. The average volume of the obtained BMA was 86.7 ± 35.2 mL. The cBMA contained a mean of 26.3 ± 6.8× 106 nucleated cells per mL, which yielded a mean of 1421.7 ± 802.7 CFUs in cell culture. There were no significant differences in NCC or number of CFUs when sex, volume of BMA, age, or BMI was examined independently (P >.05, respectively). Linear regression found that NCC was of limited predictive value for the total number of CFUs being yielded after cell culture (r2= 0.28 with a root mean square error of 679.4). NCC was of negligible predictive value for the total number of CFUs for connective tissue progenitor cells in BMA harvested from the proximal humerus during arthroscopic rotator cuff repair. NCC is often used to assess the quality of cBMA samples for biological augmentation during surgery. The limited predictive value of this measurement tool is of clinical importance, because effectiveness of BMA applications has been suggested to depend on the concentration of progenitor cells within the sample.

Analysis of Time to Form Colony Units for Connective Tissue Progenitor Cells (Stem Cells) Harvested From Concentrated Bone Marrow Aspirate and Subacromial Bursa Tissue in Patients Undergoing Rotator Cuff Repair

PurposeTo evaluate the time required for colonies to develop from concentrated bone marrow aspirate (cBMA) and subacromial bursal tissue samples.MethodsSamples of cBMA and subacromial bursa tissue were harvested from patients undergoing rotator cuff repair surgery between November 2014 and December 2019. Samples were analyzed for time to form colonies and number of colonies formed. The impact of age, sex, and cellularity (cBMA only) was analyzed. Samples were cultured and evaluated daily for colony formation in accordance with the guidelines of the International Society for Cellular Therapy. Demographic factors were analyzed for impact on time to form colonies and number of colonies formed.ResultsSamples of cBMA were obtained from 92 patients. Subacromial bursa tissue was obtained from 54 patients. For cBMA, older age was associated with more days to form colonies (P = .003), but sex (P = .955) and cellularity (P = .623) were not. For bursa, increased age was associated with longer time to form colonies (P = .002) but not sex (P = .804). Conclusions: Increased age (in cBMA and subacromial bursa tissue) and lower initial cellularity (in cBMA) are associated with longer time to form colonies in culture.Clinical RelevanceAlthough connective tissue progenitor cells are widely used in orthopaedic practice, there are few metrics to determine their efficacy. Time to form colonies may serve as an important measurement for determining connective tissue progenitor cell viability for augmentation of rotator cuff repair. Subacromial bursa tissue may represent a viable alternative to cBMA for augmentation of rotator cuff repair, capable of forming colonies expediently in vivo.

Cryopreserved bone marrow aspirate concentrate as a cell source for the colony-forming unit fibroblast assay

PurposeThe prevalence of connective tissue progenitor cells within a cell-based therapy is often quantified using the colony-forming unit fibroblast (CFU-F) assay. The present study investigates the feasibility of using cryopreserved bone marrow aspirate concentrate (BMAC) as an alternative cell source to fresh BMAC for CFU-F quantification. MethodsFreshly prepared and corresponding cryopreserved BMAC samples from patients receiving autologous cell therapy (n = 98) were analyzed using the CFU-F assay for comparison. Cultures were established by directly plating BMAC at low cell densities and maintained for a 2-week growth period. Colonies were enumerated to determine CFU-F frequency, and a subset of cultures was imaged and analyzed to quantify colony area and density. ResultsA nonlinear relationship was observed between plating density and CFU-F frequency over a wide range in plating densities (~30-fold). Cryopreserved BMAC yielded recoverable (77 ± 23%) and viable (73 ± 9%) nucleated cells upon thawing. After cryopreservation, CFU-F frequencies were found to be significantly lower (56.6 ± 34.8 vs. 50.3 ± 31.7 colonies per million nucleated cells). Yet the number of CFU-F in fresh and cryopreserved BMAC were strongly correlated (r = 0.87) and had similar area and densities. Further, moderate correlations were observed between the number of CFU-F and nucleated cells, and both the mean colony area and density were negatively correlated with patient age. Notably, no relationship was found between CFU-F frequency and age, regardless of whether fresh or cryopreserved BMAC was used. ConclusionsFreshly prepared and cryopreserved BMAC yielded correlated results when analyzed using the CFU-F assay. Our findings support the cryogenic storage of patient BMAC samples for retrospective CFU-F analyses, offering a potential alternative for characterizing BMAC and furthering our understanding of progenitor cells in relation to clinical outcome.

Clinical Outcomes Following Biologically Enhanced Patch Augmentation Repair as a Salvage Procedure for Revision Massive Rotator Cuff Tears

To evaluate the clinical outcomes of patients who underwent biologically enhanced patch augmentation repair for the treatment of revision massive rotator cuff tears. Twenty-two patients who underwent arthroscopic and mini-open rotator cuff repair using a patch augmented with platelet-rich plasma and concentrated bone marrow aspirate (cBMA) for revision massive (≥2 tendons) rotator cuff tears from 2009 to 2014, with a minimum 1-year follow-up, were included in the study. In this procedure the medial side of the graft is secured to the rotator cuff tendon remaining medially. American Shoulder and Elbow Surgeons (ASES), Simple Shoulder Test, and postoperative Single Assessment Numerical Evaluation scores were evaluated. To determine the clinical relevance of ASES scores, the minimal clinically important difference, substantial clinical benefit (SCB), and the patient-acceptable symptomatic state (PASS) thresholds were used. Clinical success or failure was defined based on whether the patient reached the SCB threshold. In thelaboratory, cellular counting along with the concentration of connective tissue progenitor cells were performed on patch samples from the day of surgery. Scaffolds were processed histologically at days 0, 7,14, and 21 of culture. Patients had significant improvement in the Simple Shoulder Test (2.6 ± 3.0pre vs 5.2 ± 4.2post, P= .01), whereas improvement in pain scores was found to be nonsignificant (5.6 ± 2.5pre vs 4.2 ± 3.4post,P= .11) at final follow-up. Mean ASES improved by Δ14.6 ± 33.4 points; however, this did not reach statistical significance (40.2 ± 21.6pre vs 53.9 ±31.4post,P= .10). With regards to ASES score, 45% of patients achieved the minimal clinically important difference, 41% the SCB, and 32% reached or exceeded the PASS criteria. At 21 days, there was a significantly greater cell count in scaffolds from patients who had clinical success than those who were failures (P= .02). Only 41% of patients undergoing biologically enhanced patch augmentation repair reached substantial clinical benefit, whereas 32% reached or exceeded the PASS criteria. Case Series: Level IV.

Connective Tissue Progenitor Analysis of Bone Marrow Aspirate Concentrate Harvested From the Body of the Ilium During Arthroscopic Acetabular Labral Repair

To evaluate the number and concentration of progenitors of the bone marrow aspirate (BMA) harvest from the body of the ilium in comparison with other established aspiration sites. The inclusion criteria consisted of primary hip arthroscopy for acetabular labral tear. BMA was performed by placing an aspiration needle into the body of ilium just proximal to the sourcil in 33 patients. The BMA was centrifuged and processed in the operating room, resulting in approximately 3 to 5 mL of bone marrow aspirate concentrate (BMAC). Samples of both BMA and BMAC sample were analyzed. The cohort of 30 patients had a mean number of nucleated cells of 24.0 million nucleated cells/cc of BMA. The BMAC samples had a mean connective tissue progenitor (CTP) cell concentration of 879.3 stem cells/cc ofBMAC, a mean CTP prevalence of 34.1 stem cells/million nucleated cells, and a mean number of days to form colonies of 2.97 days. All 4 metrics of CTP harvest did not vary significantly with age, body mass index, sex, or laterality. The nucleated cell count was significantly associated with both CTP prevalence, r2= 0.287 (P= .002), and CTP concentration, r2= 0.388 (P < .001). BMAC harvested from the body of the ilium during concurrent hip arthroscopy is a technically and biologically feasible option. Furthermore, the harvest site was found to have a CTP concentration that is similar or exceeds other published harvest sites. Finally, BMAC processing and application to areas of articular cartilage wear was performed efficiently and safely with no increase in morbidity or complications. The body of the ilium is a reliable and rich source of CTP cells. This study may assist orthopaedic surgeons interested in performing biologic augmentation during hip surgery in determining a harvest site.

Dynamic Compressive Loading Improves Cartilage Repair in an In Vitro Model of Microfracture: Comparison of 2 Mechanical Loading Regimens on Simulated Microfracture Based on Fibrin Gel Scaffolds Encapsulating Connective Tissue Progenitor Cells

Background: Microfracture of focal chondral defects often produces fibrocartilage, which inconsistently integrates with the surrounding native tissue and possesses inferior mechanical properties compared with hyaline cartilage. Mechanical loading modulates cartilage during development, but it remains unclear how loads produced in the course of postoperative rehabilitation affect the formation of the new fibrocartilaginous tissue. Purpose: To assess the influence of different mechanical loading regimens, including dynamic compressive stress or rotational shear stress, on an in vitro model of microfracture repair based on fibrin gel scaffolds encapsulating connective tissue progenitor cells. Study Design: Controlled laboratory study. Methods: Cylindrical cores were made in bovine hyaline cartilage explants and filled with either (1) cartilage plug returned to original location (positive control), (2) fibrin gel (negative control), or (3) fibrin gel with encapsulated connective tissue progenitor cells (microfracture mimic). Constructs were then subjected to 1 of 3 loading regimens: (1) no loading (ie, unloaded), (2) dynamic compressive loading, or (3) rotational shear loading. On days 0, 7, 14, and 21, the integration strength between the outer chondral ring and the central insert was measured with an electroforce mechanical tester. The central core component, mimicking microfracture neotissue, was also analyzed for gene expression by real-time reverse-transcription polymerase chain reaction, glycosaminoglycan, and double-stranded DNA contents, and tissue morphology was analyzed histologically. Results: Integration strengths between the outer chondral ring and central neotissue of the cartilage plug and fibrin + cells groups significantly increased upon exposure to compressive loading compared with day 0 controls (P = .007). Compressive loading upregulated expression of chondrogenesis-associated genes (SRY-related HGMG box-containing gene 9 [SOX9], collagen type II α1 [COL2A1], and increased ratio of COL2A1 to collagen type I α1 [COL1A1], an indicator of more hyaline phenotype) in the neotissue of the fibrin + cells group compared with the unloaded group at day 21 (SOX9, P = .0032; COL2A1, P < .0001; COL2A1:COL1A1, P = .0308). Fibrin + cells constructs exposed to shear loading expressed higher levels of chondrogenic genes compared with the unloaded condition, but the levels were not as high as those for the compressive loading condition. Furthermore, catabolic markers (MMP3 and ADAMTS 5) were significantly upregulated by shear loading (P = .0234 and P < .0001, respectively) at day 21 compared with day 0. Conclusion: Dynamic compressive loading enhanced neotissue chondrogenesis and maturation in a simulated in vitro model of microfracture, with generation of more hyaline-like cartilage and improved integration with the surrounding tissue. Clinical Relevance: Controlled loading after microfracture may be beneficial in promoting the formation of more hyaline-like cartilage repair tissue; however, the loading regimens applied in this in vitro model do not yet fully reproduce the complex loading patterns created during clinical rehabilitation. Further optimization of in vitro models of cartilage repair may ultimately inform rehabilitation protocols.

A Review of Commercially Available Point-of-Care Devices to Concentrate Bone Marrow for the Treatment of Osteoarthritis and Focal Cartilage Lesions

Mesenchymal stem cells (MSCs) are a promising cell-based therapy treatment option for several orthopedic indications. Because culture expansion of MSC is time and cost intensive, a bedside concentration of bone marrow (BM) aspirate is used as an alternative. Many commercial systems are available but the available literature and knowledge regarding these systems is limited. We compared different point-of-care devices that concentrate BM (BMC) by focusing on technical features and quality parameters to help surgeons make informed decisions while selecting the appropriate device. We compared published data on the BMC devices of Arteriocyte, Arthrex, Celling Biosciences, EmCyte, Exactech, ISTO Tech, Harvest Tech/Terumo BCT, and Zimmer/BIOMET regarding technical features (centrifugation speed/time, input/output volume, kit components, type of aspiration syringes, filter usage) and quality parameters of their final BMC product (hematocrit, concentration of platelets and total nucleated cells, concentration of MSC and connective tissue progenitor cells). The systems differ significantly in their technical features and centrifugation parameters. Only the fully automated systems use universal kits, which allow processing different volumes of BM. Only the Arthrex system allows selection of final hematocrit. There was no standardized reporting method to describe biologic potency. Based on the data obtained in this review, recommending a single device is not possible because the reported data could not be compared between devices. A standardized reporting method is needed for valid comparisons. Furthermore, clinical outcomes are required to establish the true efficacy of these systems. We are conducting additional studies for more careful comparison among the devices.

ECSIT links TLR and BMP signaling in FOP connective tissue progenitor cells.

Clinical and laboratory observations strongly suggest that the innate immune system induces flare-ups in the setting of dysregulated bone morphogenetic protein (BMP) signaling in fibrodysplasia ossificans progressiva (FOP). In order to investigate the signaling substrates of this hypothesis, we examined toll-like receptor (TLR) activation and bone morphogenetic protein (BMP) signaling in connective tissue progenitor cells (CTPCs) from FOP patients and unaffected individuals. We found that inflammatory stimuli broadly activate TLR expression in FOP CTPCs and that TLR3/TLR4 signaling amplifies BMP pathway signaling through both ligand dependent and independent mechanisms. Importantly, Evolutionarily Conserved Signaling Intermediate in the Toll Pathway (ECSIT) integrates TLR injury signaling with dysregulated BMP pathway signaling in FOP CTPCs. These findings provide novel insight into the cell autonomous integration of injury signals from the innate immune system with dysregulated response signals from the BMP signaling pathway and provide new exploratory targets for therapeutic approaches to blocking the induction and amplification of FOP lesions.