Automating iPSC generation to enable autologous photoreceptor cell replacement therapy.

BACKGROUND: Inherited retinal degeneration is a leading cause of incurable vision loss in the developed world. While autologous iPSC mediated photoreceptor cell replacement is theoretically possible, the lack of commercially available technologies designed to enable high throughput parallel production of patient specific therapeutics has hindered clinical translation. METHODS: In this study, we describe the use of the Cell X precision robotic cell culture platform to enable parallel production of clinical grade patient specific iPSCs. The Cell X is housed within an ISO Class 5 cGMP compliant closed aseptic isolator (Biospherix XVivo X2), where all procedures from fibroblast culture to iPSC generation, clonal expansion and retinal differentiation were performed. RESULTS: Patient iPSCs generated using the Cell X platform were determined to be pluripotent via score card analysis and genetically stable via karyotyping. As determined via immunostaining and confocal microscopy, iPSCs generated using the Cell X platform gave rise to retinal organoids that were indistinguishable from organoids derived from manually generated iPSCs. In addition, at 120 days post-differentiation, single-cell RNA sequencing analysis revealed that cells generated using the Cell X platform were comparable to those generated under manual conditions in a separate laboratory. CONCLUSION: We have successfully developed a robotic iPSC generation platform and standard operating procedures for production of high-quality photoreceptor precursor cells that are compatible with current good manufacturing practices. This system will enable clinical grade production of iPSCs for autologous retinal cell replacement.

What is the Efficacy of Intra-Articular Platelet-Rich Plasma Injections for Knee Osteoarthritis in Clinical Practice? A "Real-Life" Prospective Cohort.

INTRODUCTION: Platelet-rich plasma (PRP) injections may improve symptoms in patients suffering from knee osteoarthritis. However, there is a lack of data on its effectiveness in a "real-life" cohort. This multi-site institutional registry study aimed to assess patients' longitudinal progress after PRP injection for knee osteoarthritis. MATERIALS AND METHODS: All patients receiving PRP injections for knee osteoarthritis at a large, integrated tertiary academic center (December 18, 2017 to March 1, 2021) were eligible. A prospective data collection instrument was used to collect patient demographics, procedural information, and patient-reported outcome measures. Overall, 97 patients met the inclusion criteria, and 53 were included in the analysis. RESULTS: One in four patients (26%) improved on all three Knee Injury and Osteoarthritis Outcome Score subscales: 17% in two subscales and 20% in one subscale, respectively. Overall, 64% of patients improved in at least one patient-reported outcomes measure. At six months post injection, 49% of patients were satisfied. CONCLUSION: PRP injection provides positive changes in two out of three patients in different magnitudes and characteristics with careful attention to clinically meaningful differences.

Characterization of heterogeneous primary human cartilage-derived cell population using non-invasive live-cell phase-contrast time-lapse imaging.

Reliable and reproducible cell therapy strategies to treat osteoarthritis demand an improved characterization of the cell and heterogeneous cell population resident in native cartilage tissue. Using live-cell phase-contrast time-lapse imaging (PC-TLI), this study investigates the morphological attributes and biological performance of the three primary biological objects enzymatically isolated from primary human cartilage: connective tissue progenitors (CTPs), non-progenitors (NPs) and multi-cellular structures (MCSs). The authors' results demonstrated that CTPs were smaller in size in comparison to NPs (P < 0.001). NPs remained part of the adhered cell population throughout the cell culture period. Both NPs and CTP progeny on day 8 increased in size and decreased in circularity in comparison to their counterparts on day 1, although the percent change was considerably less in CTP progeny (P < 0.001). PC-TLI analyses indicated three colony types: single-CTP-derived (29%), multiple-CTP-derived (26%) and MCS-derived (45%), with large heterogeneity with respect to cell morphology, proliferation rate and cell density. On average, clonal (CL) (P = 0.009) and MCS (P = 0.001) colonies exhibited higher cell density (cells per colony area) than multi-clonal (MC) colonies; however, it is interesting to note that the behavior of CL (less cells per colony and less colony area) and MCS (high cells per colony and high colony area) colonies was quite different. Overall effective proliferation rate (EPR) of the CTPs that formed CL colonies was higher than the EPR of CTPs that formed MC colonies (P = 0.02), most likely due to CTPs with varying EPR that formed the MC colonies. Finally, the authors demonstrated that lag time before first cell division of a CTP (early attribute) could potentially help predict its proliferation rate long-term. Quantitative morphological characterization using non-invasive PC-TLI serves as a reliable and reproducible technique to understand cell heterogeneity. Size and circularity parameters can be used to distinguish CTP from NP populations. Morphological cell and colony features can also be used to reliably and reproducibly identify CTP subpopulations with preferred proliferation and differentiation potentials in an effort to improve cell manufacturing and therapeutic outcomes.

The Potential Effects of Imposing a Body Mass Index Threshold on Patient-Reported Outcomes After Total Knee Arthroplasty.

BACKGROUND: Operative eligibility thresholds based on body mass index (BMI) alone may risk restricting access to improved pain control, function, and quality of life. This study evaluated the use of BMI-cutoffs to offering TKA in avoiding: 1) 90-day readmission, 2) one-year mortality, and 3) failure to achieve clinically important one-year PROMS improvement (MCID). METHODS: A total of 4126 primary elective unilateral TKA patients from 2015 to 2018 were prospectively collected. For specific BMI(kg/m2) cutoffs: 30, 35, 40, 45, and 50, the positive predictive value (PPV) for 90-day readmission, one-year mortality, and failure to achieve one-year MCID were calculated. The number of patients denied complication-free postoperative courses per averted adverse outcome/failed improvement was estimated. RESULTS: Rates of 90-day readmission and one-year mortality were similar across BMI categories (P > .05, each). PPVs for preventing 90-day readmission and one-year mortality were low across all models of BMI cutoffs. The highest PPV for 90-day readmission and one-year mortality was detected at cutoffs of 45 (6.4%) and 40 (0.87%), respectively. BMI cutoff of 40 would deny 18 patients 90-day readmission-free, and 194 patients one-year mortality-free postoperative courses for each averted 90-day readmission/one-year mortality. Such cutoff would also deny 11 patients an MCID per avoided failure. Implementing BMI thresholds alone did not influence the rate of improvements in KOOS-PS, KRQOL, or VR-12. CONCLUSION: Utilizing BMI cutoffs as the sole determinants of TKA ineligibility may deny patients complication-free postoperative courses and clinically important improvements. Shared decision-making supported by predictive tools may aid in balancing the potential benefit TKA offers to obese patients with the potentially increased complication risk and cost of care provision.

Variation in primary and culture-expanded cells derived from connective tissue progenitors in human bone marrow space, bone trabecular surface and adipose tissue.

BACKGROUND AIMS: Connective tissue progenitors (CTPs) embody the heterogeneous stem and progenitor cell populations present in native tissue. CTPs are essential to the formation and remodeling of connective tissue and represent key targets for tissue-engineering and cell-based therapies. To better understand and characterize CTPs, we aimed to compare the (i) concentration and prevalence, (ii) early in vitro biological behavior and (iii) expression of surface-markers and transcription factors among cells derived from marrow space (MS), trabecular surface (TS), and adipose tissues (AT). METHODS: Cancellous-bone and subcutaneous-adipose tissues were collected from 8 patients. Cells were isolated and cultured. Colony formation was assayed using Colonyze software based on ASTM standards. Cell concentration ([Cell]), CTP concentration ([CTP]) and CTP prevalence (PCTP) were determined. Attributes of culture-expanded cells were compared based on (i) effective proliferation rate and (ii) expression of surface-markers CD73, CD90, CD105, SSEA-4, SSEA-3, SSEA-1/CD15, Cripto-1, E-Cadherin/CD324, Ep-CAM/CD326, CD146, hyaluronan and transcription factors Oct3/4, Sox-2 and Nanog using flow cytometry. RESULTS: Mean [Cell], [CTP] and PCTP were significantly different between MS and TS samples (P = 0.03, P = 0.008 and P= 0.0003), respectively. AT-derived cells generated the highest mean total cell yield at day 6 of culture-4-fold greater than TS and more than 40-fold greater than MS per million cells plated. TS colonies grew with higher mean density than MS colonies (290 ± 11 versus 150 ± 11 cell per mm2; P = 0.0002). Expression of classical-mesenchymal stromal cell (MSC) markers was consistently recorded (>95%) from all tissue sources, whereas all the other markers were highly variable. CONCLUSIONS: The prevalence and biological potential of CTPs are different between patients and tissue sources and lack variation in classical MSC markers. Other markers are more likely to discriminate differences between cell populations in biological performance. Understanding the underlying reasons for variation in the concentration, prevalence, marker expression and biological potential of CTPs between patients and source tissues and determining the means of managing this variation will contribute to the rational development of cell-based clinical diagnostics and targeted cell-based therapies.

Proceedings of the signature series symposium "cellular therapies for orthopaedics and musculoskeletal disease proven and unproven therapies-promise, facts and fantasy," international society for cellular therapies, montreal, canada, may 2, 2018.

The Signature Series Symposium "Cellular Therapies for Orthopaedics and Musculoskeletal Disease Proven and Unproven Therapies-Promise, Facts and Fantasy" was held as a pre-meeting of the 26th International Society for Cellular Therapy (ISCT) annual congress in Montreal, Canada, May 2, 2018. This was the first ISCT program that was entirely dedicated to the advancement of cell-based therapies for musculoskeletal diseases. Cellular therapies in musculoskeletal medicine are a source of great promise and opportunity. They are also the source of public controversy, confusion and misinformation. Patients, clinicians, scientists, industry and government share a commitment to clear communication and responsible development of the field. Therefore, this symposium convened thought leaders from around the world in a forum designed to catalyze communication and collaboration to bring the greatest possible innovation and value to patients with musculoskeletal conditions.

High occurrence of osteoarthritic histopathological features unaccounted for by traditional scoring systems in lateral femoral condyles from total knee arthroplasty patients with varus alignment.

Background and purpose - A better understanding of the patterns and variation in initiation and progression of osteoarthritis (OA) in the knee may influence the design of therapies to prevent or slow disease progression. By studying cartilage from the human lateral femoral condyle (LFC), we aimed to: (1) assess specimen distribution into early, mild, moderate, and severe OA as per the established histopathological scoring systems (HHGS and OARSI); and (2) evaluate whether these 2 scoring systems provide sufficient tools for characterizing all the features and variation in patterns of OA. Patients and methods - 2 LFC osteochondral specimens (4 x 4 x 8 mm) were collected from 50 patients with idiopathic OA varus knee and radiographically preserved lateral compartment joint space undergoing total knee arthroplasty. These were fixed, sectioned, and stained with HE and Safranin O-Fast Green (SafO). Results - The histopathological OA severity distribution of the 100 specimens was: 6 early, 62 mild, 30 moderate, and 2 severe. Overall, 45/100 specimens were successfully scored by both HHGS and OARSI: 12 displayed low OA score and 33 displayed cartilage surface changes associated with other histopathological features. However, 55/100 samples exhibited low surface structure scores, but were deemed to be inadequately scored by HHGS and OARSI because of anomalous features in the deeper zones not accounted for by these systems: 27 exhibited both SafO and tidemark abnormal features, 16 exhibited only SafO abnormal features, and 12 exhibited tidemark abnormal features. Interpretation - LFC specimens were scored as mild to moderate OA by HHGS and OARSI. Yet, several specimens exhibited deep zone anomalies while maintaining good surface structure, inconsistent with mild OA. Overall, a better classification of these anomalous histopathological features could help better understand idiopathic OA and potentially recognize different subgroups of disease.

Stem cell industry update: 2012 to 2016 reveals accelerated investment, but market capitalization and earnings lag.

Treatments based on stem cells have long been heralded for their potential to drive the future of regenerative medicine and have inspired increasing medical and business interest. The stem cell therapy market has been expanding since 2012, but earnings and profitability still lag the broader health care sector (compounded annual growth rate in annual financing of 31.5% versus 13.4%, respectively). On the basis of historical financial data, approximately $23 billion has been invested in stem cell companies since 1994, with more than 80% of this raised from 2011 through 2016. This reflects a marked acceleration in capital investment, as companies began late-stage clinical trials, initiate partnerships or are acquired by large pharmaceutical companies. All of these data reflect a field that is emerging from infancy, which will demand more time and capital to mature. This update is relevant to researchers, clinicians and investors who wish to quantify the potential in this field.

Cellular therapy injections in today's orthopedic market: A social media analysis.

BACKGROUND: The current state of cellular therapy for musculoskeletal conditions is at a crossroads. Marketing efforts are often outpacing clinical evidence and regulatory control. QUESTIONS/PURPOSES: This study was an effort to describe the marketing of cellular therapy in musculoskeletal medicine by evaluating the content in popular social media channels. Specifically, media posts were evaluated for the following: (1) perspective, (2) tone, (3) content and (4) visibility. PATIENT AND METHODS: Social media content related to cell therapy for musculoskeletal conditions was assessed in a search using 28 hashtags on the public domains of Instagram and Twitter over a 2-year period (2014-2016) that resulted in analysis of 698 posts. Supplemental analyses of LinkedIn and Facebook domains were also conducted. A categorical scoring system was used to analyze perspective (patient, family or friend, business or organization), tone (positive, negative), content (education, advertisement, research, media coverage or patient experience) and visibility (number of hashtags per post). Sub-analyses of the advertisement content from various perspectives (patients, physicians and businesses) were performed. RESULTS: The media perspective was most frequently from a business or organization (83%; n = 575). A total of 94% of the posts had a positive tone and only 6% had a negative tone, and the only negative posts came from patients (60% positive and 40% negative). The most common content of social media posts were advertisements, representing 68% (n = 477) of all posts; this was confirmed in the Facebook analysis. The mean number of hashtags was five per post. Sub-analyses revealed approximately half of the advertising posts originated from a single business that recruited physicians to market their cell-based therapies on social media, which was confirmed in the LinkedIn analysis. CONCLUSION: The market messages related to cell-based therapies for musculoskeletal conditions available on social media are dominated by businesses that seem to use a network of physicians, apply several hashtags to enhance visibility and advertise these largely unproven modalities. The posts portray an almost exclusively positive tone, without providing a "fair balance" on the risks, benefits and limitations.

The Effect of Surgical Technique and Spacer Texture on Bone Regeneration: A Caprine Study Using the Masquelet Technique.

BACKGROUND: The Masquelet-induced-membrane technique is a commonly used method for treating segmental bone defects. However, there are no established clinical standards for management of the induced membrane before grafting. QUESTIONS/PURPOSES: Two clinically based theories were tested in a chronic caprine tibial defect model: (1) a textured spacer that increases the induced-membrane surface area will increase bone regeneration; and (2) surgical scraping to remove a thin tissue layer of the inner induced-membrane surface will enhance bone formation. METHODS: Thirty-two skeletally mature female goats were assigned to four groups: smooth spacer with or without membrane scraping and textured spacer with or without membrane scraping. During an initial surgical procedure (unilateral, left tibia), a defect was created excising bone (5 cm), periosteum (9 cm), and muscle (10 g). Segments initially were stabilized with an intramedullary rod and an antibiotic-impregnated polymethylmethacrylate spacer with a smooth or textured surface. Four weeks later, the spacer was removed and the induced-membrane was either scraped or left intact before bone grafting. Bone formation was assessed using micro-CT (total bone volume in 2.5-cm central defect region) as the primary outcome; radiographs and histologic analysis as secondary outcomes, with the reviewer blinded to the treatment groups of the samples being assessed 12 weeks after grafting. All statistical tests were performed using a linear mixed effects model approach. RESULTS: Micro-CT analysis showed greater bone formation in defects with scraped induced membrane (mean, 3034.5 mm3; median, 1928.0 mm3; quartile [Q]1-Q3, 273.3-2921.1 mm3) compared with defects with intact induced membrane (mean, 1709.5 mm3; median, 473.8 mm3; Q1-Q3, 132.2-1272.3 mm3; p = 0.034). There was no difference in bone formation between textured spacers (mean, 2405.5 mm3; median, 772.7 mm3; Q1-Q3, 195.9-2743.8 mm3) and smooth spacers (mean, 2473.2 mm3; median, 1143.6 mm3; Q1-Q3, 230.2-451.1 mm3; p = 0.917). CONCLUSIONS: Scraping the induced-membrane surface to remove the innermost layer of the induced-membrane increased bone regeneration. A textured spacer that increased the induced-membrane surface area had no effect on bone regeneration. CLINICAL RELEVANCE: Scraping the induced membrane during the second stage of the Masquelet technique may be a rapid and simple means of improving healing of segmental bone defects, which needs to be confirmed clinically.

Open mHealth Architecture: A Primer for Tomorrow's Orthopedic Surgeon and Introduction to Its Use in Lower Extremity Arthroplasty.

BACKGROUND: The recent private-public partnership to unlock and utilize all available health data has large-scale implications for public health and personalized medicine, especially within orthopedics. Today, consumer based technologies such as smartphones and "wearables" store tremendous amounts of personal health data (known as "mHealth") that, when processed and contextualized, have the potential to open new windows of insight for the orthopedic surgeon about their patients. METHODS: In the present report, the landscape, role, and future technical considerations of mHealth and open architecture are defined with particular examples in lower extremity arthroplasty. RESULTS: A limitation of the current mHealth landscape is the fragmentation and lack of interconnectivity between the myriad of available apps. The importance behind the currently lacking open mHealth architecture is underscored by the offer of improved research, increased workflow efficiency, and value capture for the orthopedic surgeon. CONCLUSION: There exists an opportunity to leverage existing mobile health data for orthopaedic surgeons, particularly those specializing in lower extremity arthroplasty, by transforming patient small data into insightful big data through the implementation of "open" architecture that affords universal data standards and a global interconnected network.

Evidence for the Use of Cell-Based Therapy for the Treatment of Osteonecrosis of the Femoral Head: A Systematic Review of the Literature.

BACKGROUND: Cell-therapy has been promoted among the therapeutic arsenal that can aid in bone formation and remodeling, in early stages of osteonecrosis of the femoral head (ONFH). The purpose of this systematic review was to assess the evidence supporting the (1) clinical efficacy; (2) structural modifying effect, as evaluated radiographically; (3) revision rates; and (4) safety of cell-therapy for the treatment of ONFH. METHODS: A systematic review was performed including studies with a level-of-evidence of III or higher. A total of 1483 articles were screened. Eleven studies met the criteria for inclusion in this review (level-of-evidence: 6 level-I, 1 level-II, and 4 level-III), including 683 cases of ONFH. RESULTS: All 10 studies that reported patient-reported outcomes showed improved outcomes in the cell-therapy groups compared with the control group. Overall, 24.5% (93/380 hips) that received cell-therapy showed radiographic progression compared with 40% (98/245 hips) in the control group. Nine of 10 studies that reported failure rates showed a lower total hip arthroplasty conversion rate in the cell-therapy group 16% (62/380 hips) compared with the control group 21% (52/252 hips). There was a low complication rate (<3%) with no major adverse effects. CONCLUSION: Cell-therapies for the treatment of ONFH have been reported to be safe and suggest improved clinical outcomes with lower disease progression rate. However, there was substantial heterogeneity in the included studies, and in the cell-based therapies used. Specific clinical indications and cell-therapy standardization are required because studies varied widely with respect to cell sourcing, cell characterization, adjuvant therapies, and assessment of outcomes.

Analysis of Cell Therapies Used in Clinical Trials for the Treatment of Osteonecrosis of the Femoral Head: A Systematic Review of the Literature.

BACKGROUND: Osteonecrosis of the femoral head (ONFH) is associated with regional loss of cells within bone, often resulting in pain and mechanical collapse. Our purpose was to analyze the cell-therapies used in clinical trials for the treatment of ONFH with regard to (1) cell-sources, (2) collection techniques, (3) cell-processing, (4) qualitative and quantitative characterizations, and (5) delivery methods. METHODS: A systematic review of the current literature on the use of cell therapies for the treatment of ONFH was performed. Studies with a level-of-evidence III or higher were evaluated. A total of 1483 articles were screened. Eleven studies met the criteria to be included in this review. RESULTS: Ten studies used bone-marrow, and 1 study used blood as the cell-source. Nine studies used freshly isolated tissue-derived nucleated cells from bone-marrow, mixed bone marrow-derived nucleated cells, 1 study used mixed blood-derived nucleated cells, and 1 study used culture-expanded cells derived from bone marrow aspirate. Cell dose varied from 2-million to 3-billion cells. Qualitative cell characterization of injected cells using surface markers was done by 5 studies using CD34. Two studies assayed the cell-population using a colony-forming-unit assay. CONCLUSION: There is a lack of standardization with respect to the quantitative and qualitative characterization of methods for cell-harvest, cell-processing, and cell-transplantation/delivery. Cell-therapy holds promise as a means of restoring local cell populations that are made deficient because of injury or disease. However, the orthopedic community and patients will benefit greatly by a greater investment in blinded, randomized, controlled trials and clinical effectiveness trials that embrace rigorous standards.

Early Termination of Randomized Clinical Trials in Orthopaedics.

Randomized controlled clinical trials (RCTs) in orthopaedics, similar to all medical fields, provide the highest level of clinical data and constitute the cornerstone for evidence-based therapeutic advances. Orthopaedic clinical trials face the same challenges as in other medical fields, such as insufficient recruitment, unforeseen adverse events, and futility. In this article, we highlight the reasons for early termination of clinical trials and provide examples of each type.

Cellular Therapies in Orthopedics: Where Are We?

The orthopedic field has experienced several major practice-changing pivotal shifts in the past several decades, such as the invention and application of the arthroscope or the implementation and advancement of joint arthroplasties. Most of these previous breakthroughs have focused on surgical techniques and devices. However, the next major advance in the field is likely to be related to biologic treatments. Although still in its early stage of development, orthopedic regenerative medicine, including cellular therapies, represents a great opportunity, since we are only beginning to understand their biological potential. The main challenge in this pathway is to translate the promising results obtained by basic scientists to clinical practice. This work reviewed the market and clinical evidence, as well as future perspectives, concerning cellular therapies in orthopedics.

What Influences How Patients Rate Their Hospital Stay After Total Hip Arthroplasty?

PURPOSE: Measuring the effect of operative interventions has been a challenge in orthopaedics. The assessment of patient satisfaction, as measured by the Press Ganey (PG) satisfaction surveys, has gained increasing attention. Our purpose was to determine the factors and patient characteristics that influence patient satisfaction after THA. MATERIALS AND METHODS: The PG database was queried identifying 692 THA patients (November 2009 to January 2015). A multiple regression analysis was conducted. RESULTS: Significant influence was found in communication with nurses (p=<0.001), response time of hospital staff (p=0.001), communication with physicians (p=0.002), and hospital environment (p=0.049). Management of pain and communication about medications were not significant for overall hospital rating. There were no differences between PG scores of patients who did and did not have complications. CONCLUSIONS: Hospital rating was significantly influenced by patients' communication with nurses, response time of hospital staff, and communication with physicians. Recognizing the importance of these elements, can greatly improve patient satisfaction after THA.

What Influences How Patients with Depression Rate Hospital Stay After Total Joint Arthroplasty?

BACKGROUND: Recent healthcare reform has spurred important changes to provider reimbursement. With the implementation of the Value Based Purchasing program, significant weight is placed on patient experience of care. The Press Ganey (PG) survey is currently used by over 10,000 hospitals, as it serves to help optimize patient satisfaction. However, confounding factors, such as clinical depression, are not screened against by PG. Thus, arthroplasty surgeons performing lower extremity total joint arthroplasty (TJA) may have difficulty optimizing patient satisfaction while caring for patients with clinical depression. Therefore, we asked: 1) What Press Ganey elements affect the overall hospital rating in patients who suffer from clinical depression? and 2) Are survey responses different between patients who do and do not have clinical depression? MATERIALS AND METHODS: We queried our institutional PG database for patients who underwent a TJA from November 2009 to January 2015. Our search yielded 1,454 patients, of which 204 suffered from depression and 1,250 did not. Multiple regression analysis was performed to determine the influence (b weight) of selected PG survey domains on overall hospital rating. The weighted mean for domain was also calculated. RESULTS: Multiple regression analyses showed that overall hospital ratings were significantly influenced by communication with nurses (b-weight = 0.881, p< 0.001) in post-TJA patients with depression. The remaining domains were not statistically significant. There were no significant differences in individual PG elements for patients who did and did not have depression. CONCLUSION: Overall patient satisfaction among patients with depression was greatly influenced by communication with nurses. Understanding these challenges may encourage care coordination across disciplines for the management of patients with depression before and after surgery. As a result, this could optimize orthopedic surgery outcomes, but, more importantly, patient health and satisfaction, while reducing costs of care.

Circular Halbach array for fast magnetic separation of hyaluronan-expressing tissue progenitors.

Connective tissue progenitors (CTPs) are a promising therapeutic agent for bone repair. Hyaluronan, a high molecular mass glycosaminoglycan, has been shown by us to be a suitable biomarker for magnetic separation of CTPs from bone marrow aspirates in a canine model. For the therapy to be applicable in humans, the magnetic separation process requires scale-up without compromising the viability of the cells. The scaled-up device presented here utilizes a circular Halbach array of diametrically magnetized, cylindrical permanent magnets. This allows precise control of the magnetic field gradient driving the separation, with theoretical analysis favoring a hexapole field. The separation vessel has the external diameter of a 50 mL conical centrifuge tube and has an internal rod that excludes cells from around the central axis. The magnet and separation vessel (collectively dubbed the hexapole magnet separator or HMS) was tested on four human and four canine bone marrow aspirates. Each CTP-enriched cell product was tested using cell culture bioassays as surrogates for in vivo engraftment quality. The magnetically enriched cell fractions showed statistically significant, superior performance compared to the unenriched and depleted cell fractions for all parameters tested, including CTP prevalence (CTPs per 10(6) nucleated cells), proliferation by colony forming unit (CFU) counts, and differentiation by staining for the presence of osteogenic and chondrogenic cells. The simplicity and speed of the HMS operation could allow both CTP isolation and engraftment during a single surgical procedure, minimizing trauma to patients and lowering cost to health care providers.

Response of bone marrow derived connective tissue progenitor cell morphology and proliferation on geometrically modulated microtextured substrates.

Varying geometry and layout of microposts on a cell culture substrate provides an effective technique for applying mechanical stimuli to living cells. In the current study, the optimal geometry and arrangement of microposts on the polydimethylsiloxane (PDMS) surfaces to enhance cell growth behavior were investigated. Human bone marrow derived connective tissue progenitor cells were cultured on PDMS substrates comprising unpatterned smooth surfaces and cylindrical post microtextures that were 10 µm in diameter, 4 heights (5, 10, 20 and 40 µm) and 3 pitches (10, 20, and 40 µm). With the same 10 µm diameter, post heights ranging from 5 to 40 µm resulted in a more than 535 fold range of rigidity from 0.011 nNµm⁻¹ (40 µm height) up to 5.888 nNµm⁻¹(5 µm height). Even though shorter microposts result in higher effective stiffness, decreasing post heights below the optimal value, 5 µm height micropost in this study decreased cell growth behavior. The maximum number of cells was observed on the post microtextures with 20 µm height and 10 µm inter-space, which exhibited a 675 % increase relative to the smooth surfaces. The cells on all heights of post microtextures with 10 µm and 20 µm inter-spaces exhibited highly contoured morphology. Elucidating the cellular response to various external geometry cues enables us to better predict and control cellular behavior. In addition, knowledge of cell response to surface stimuli could lead to the incorporation of specific size post microtextures into surfaces of implants to achieve surface-textured scaffold materials for tissue engineering applications.

Tibial Bushing Wear at 28 Years of Follow-up After Rotating-Hinge Distal Femoral Replacement: A Case Report.

CASE: A 58-year-old woman presented with swelling, stiffness, and pain of the right knee 28 years after rotating-hinge distal femoral replacement after osteosarcoma resection. She underwent revision. There was wear through the entire thickness of the polyethylene tibial sleeve bushing, and the implant was well-fixed. The knee was reassembled with new bushings, sleeves, yoke, axle, poly, and a locking pin. CONCLUSION: This is the first described case of tibial sleeve bushing wear. It highlights the importance of early detection of potential complications and implant surveillance because it can enable surgeons to intervene with minor procedures, avoiding eventual catastrophic failure.