A miniaturized culture platform for control of the metabolic environment.

The heart is a metabolic "omnivore" and adjusts its energy source depending on the circulating metabolites. Human cardiac organoids, a three-dimensional in vitro model of the heart wall, are a useful tool to study cardiac physiology and pathology. However, cardiac tissue naturally experiences shear stress and nutrient fluctuations via blood flow in vivo, whilst in vitro models are conventionally cultivated in a static medium. This necessitates the regular refreshing of culture media, which creates acute cellular disturbances and large metabolic fluxes. To culture human cardiac organoids in a more physiological manner, we have developed a perfused bioreactor for cultures in a 96-well plate format. The designed bioreactor is easy to fabricate using a common culture plate and a 3D printer. Its open system allows for the use of traditional molecular biology techniques, prevents flow blockage issues, and provides easy access for sampling and cell assays. We hypothesized that a perfused culture would create more stable environment improving cardiac function and maturation. We found that lactate is rapidly produced by human cardiac organoids, resulting in large fluctuations in this metabolite under static culture. Despite this, neither medium perfusion in bioreactor culture nor lactate supplementation improved cardiac function or maturation. In fact, RNA sequencing revealed little change across the transcriptome. This demonstrates that cardiac organoids are robust in response to fluctuating environmental conditions under normal physiological conditions. Together, we provide a framework for establishing an easily accessible perfusion system that can be adapted to a range of miniaturized cell culture systems.

Orthopedic Training in the United States A Continuously Evolving Process.

Orthopedic surgery in the United States has gone through many changes over the past few centuries. Starting with a small sect of subspecialized surgeons, advances in technology and surgical skills have paralleled the growth of the specialty. To keep up with demand, the training of orthopedic surgeons has undergone many iterations. From apprenticeships to the current residency model, the field has always adapted to ensure the constant production of well-trained surgeons to take care of the growing orthopedic needs in the population. In order to guarantee this, many regulatory committees have been formed over the years to help guide the regulation and certification of orthopedic training programs. With current day residents facing new challenges, the specialty continues to adapt the way it trains its future.

A tumour-spheroid manufacturing and cryopreservation process that yields a highly reproducible product ready for direct use in drug screening assays.

If it were possible to purchase tumour-spheroids as a standardised product, ready for direct use in assays, this may contribute to greater research reproducibility, potentially reducing costs and accelerating outcomes. Herein, we describe a workflow where uniformly sized cancer tumour-spheroids are mass-produced using microwell culture, cryopreserved with high viability, and then cultured in neutral buoyancy media for drug testing. C4-2B prostate cancer or MCF-7 breast cancer cells amalgamated into uniform tumour-spheroids after 48 h of culture. Tumour-spheroids formed from 100 cells each tolerated the cryopreservation process marginally better than tumour-spheroids formed from 200 or 400 cells. Post-thaw, tumour-spheroid metabolic activity was significantly reduced, suggesting mitochondrial damage. Metabolic function was rescued by thawing the tumour-spheroids into medium supplemented with 10 µM N-Acetyl-l-cysteine (NAC). Following thaw, the neutral buoyancy media, Happy Cell ASM, was used to maintain tumour-spheroids as discrete tissues during drug testing. Fresh and cryopreserved C4-2B or MCF-7 tumour-spheroids responded similarly to titrations of Docetaxel. This protocol will contribute to a future where tumour-spheroids may be available for purchase as reliable and reproducible products, allowing laboratories to efficiently replicate and build on published research, in many cases, making tumour-spheroids simply another cell culture reagent.

Method for manufacture and cryopreservation of cartilage microtissues.

The financial viability of a cell and tissue-engineered therapy may depend on the compatibility of the therapy with mass production and cryopreservation. Herein, we developed a method for the mass production and cryopreservation of 3D cartilage microtissues. Cartilage microtissues were assembled from either 5000 human bone marrow-derived stromal cells (BMSC) or 5000 human articular chondrocytes (ACh) each using a customized microwell platform (the Microwell-mesh). Microtissues rapidly accumulate homogenous cartilage-like extracellular matrix (ECM), making them potentially useful building blocks for cartilage defect repair. Cartilage microtissues were cultured for 5 or 10 days and then cryopreserved in 90% serum plus 10% dimethylsulfoxide (DMSO) or commercial serum-free cryopreservation media. Cell viability was maximized during thawing by incremental dilution of serum to reduce oncotic shock, followed by washing and further culture in serum-free medium. When assessed with live/dead viability dyes, thawed microtissues demonstrated high viability but reduced immediate metabolic activity relative to unfrozen control microtissues. To further assess the functionality of the freeze-thawed microtissues, their capacity to amalgamate into a continuous tissue was assess over a 14 day culture. The amalgamation of microtissues cultured for 5 days was superior to those that had been cultured for 10 days. Critically, the capacity of cryopreserved microtissues to amalgamate into a continuous tissue in a subsequent 14-day culture was not compromised, suggesting that cryopreserved microtissues could amalgamate within a cartilage defect site. The quality ECM was superior when amalgamation was performed in a 2% O2 atmosphere than a 20% O2 atmosphere, suggesting that this process may benefit from the limited oxygen microenvironment within a joint. In summary, cryopreservation of cartilage microtissues is a viable option, and this manipulation can be performed without compromising tissue function.

SP7 gene silencing dampens bone marrow stromal cell hypertrophy, but it also dampens chondrogenesis.

For bone marrow stromal cells (BMSC) to be useful in cartilage repair their propensity for hypertrophic differentiation must be overcome. A single day of TGF-ß1 stimulation activates intrinsic signaling cascades in BMSCs which subsequently drives both chondrogenic and hypertrophic differentiation. TGF-ß1 stimulation upregulates SP7, a transcription factor known to contribute to hypertrophic differentiation, and SP7 remains upregulated even if TGF-ß1 is subsequently withdrawn from the chondrogenic induction medium. Herein, we stably transduced BMSCs to express an shRNA designed to silence SP7, and assess the capacity of SP7 silencing to mitigate hypertrophy. SP7 silencing dampened both hypertrophic and chondrogenic differentiation processes, resulting in diminished microtissue size, impaired glycosaminoglycan production and reduced chondrogenic and hypertrophic gene expression. Thus, while hypertrophic features were dampened by SP7 silencing, chondrogenic differentation was also compromised. We further investigated the role of SP7 in monolayer osteogenic and adipogenic cultures, finding that SP7 silencing dampened characteristic mineralization and lipid vacuole formation, respectively. Overall, SP7 silencing affects the trilineage differentiation of BMSCs, but is insufficient to decouple BMSC hypertrophy from chondrogenesis. These data highlight the challenge of promoting BMSC chondrogenesis whilst simultaneously reducing hypertrophy in cartilage tissue engineering strategies.

Polyethylene liner dissociation from humeral tray: impediment to closed reduction of dislocated reverse total shoulder replacement.

Background: Instability is one of the leading causes of revision for reverse total shoulder arthroplasty (RTSA). Closed reduction (CR) of a dislocated RTSA is recommended by many as initial treatment with varying degrees of success. In this study, we describe polyethylene liner dissociation from the humeral tray (PDH) as a cause of failure of CR of dislocated RTSA. Methods: In this retrospective study, patients who underwent revision RTSA for instability were identified through our institutional database review using specific International Classification of Diseases and Current Procedural Terminology codes. Pertinent clinical information including demographics, details of instability event (early vs. late), traumatic vs. atraumatic, outcomes of CR (if performed), and intraoperative findings during revision surgery were collected and analyzed. Results: Twenty-two patients met the inclusion criteria with average follow-up of 2 years. CR was attempted in 12 (55%) patients, prior to revision surgery, and was successful in 5 (23%) patients. During the revision surgery polyethylene liner dissociation from the humeral tray (PDH) was identified in 10 patients (45%). Five of these 10 patients had failed CR and the other 5 patients did not undergo CR due to primary surgeon's preference. All patients with PDH event had onlay humeral tray RTSA system. Although not a consistent radiographic finding in our series, the presence of the metallic glenosphere in direct contact with the humeral tray on anteroposterior or axillary radiographs was diagnostic for PDH. Conclusion: Dissociation of polyethylene liner from the humeral tray can be associated with an RTSA dislocation and is a contraindication for CR. A radiographic finding of the metallic humeral tray articulating directly with the glenosphere is an indication that the polyethylene liner is dissociated from the humeral tray.

Spray nebulization enables polycaprolactone nanofiber production in a manner suitable for generation of scaffolds or direct deposition of nanofibers onto cells.

Spray nebulization is an elegant, but relatively unstudied, technique for scaffold production. Herein we fabricated mesh scaffolds of polycaprolactone (PCL) nanofibers via spray nebulization of 8% PCL in dichloromethane (DCM) using a 55.2 kPa compressed air stream and 17 ml h-1polymer solution flow rate. Using a refined protocol, we tested the hypothesis that spray nebulization would simultaneously generate nanofibers and eliminate solvent, yielding a benign environment at the point of fiber deposition that enabled the direct deposition of nanofibers onto cell monolayers. Nanofibers were collected onto a rotating plate 20 cm from the spray nozzle, but could be collected onto any static or moving surface. Scaffolds exhibited a mean nanofiber diameter of 910 ± 190 nm, ultimate tensile strength of 2.1 ± 0.3 MPa, elastic modulus of 3.3 ± 0.4 MPa, and failure strain of 62 ± 6%.In vitro, scaffolds supported growth of human keratinocyte cell epithelial-like layers, consistent with potential utility as a dermal scaffold. Fourier-transform infrared spectroscopy demonstrated that DCM had vaporized and was undetectable in scaffolds immediately following production. Exploiting the rapid elimination of DCM during fiber production, we demonstrated that nanofibers could be directly deposited on to cell monolayers, without compromising cell viability. This is the first description of spray nebulization generating nanofibers using PCL in DCM. Using this method, it is possible to rapidly produce nanofiber scaffolds, without need for high temperatures or voltages, yielding a method that could potentially be used to deposit nanofibers onto cell cultures or wound sites.

Microtissue Culture Provides Clarity on the Relative Chondrogenic and Hypertrophic Response of Bone-Marrow-Derived Stromal Cells to TGF-ß1, BMP-2, and GDF-5.

Chondrogenic induction of bone-marrow-derived stromal cells (BMSCs) is typically accomplished with medium supplemented with growth factors (GF) from the transforming growth factor-beta (TGF-ß)/bone morphogenetic factor (BMP) superfamily. In a previous study, we demonstrated that brief (1-3 days) stimulation with TGF-ß1 was sufficient to drive chondrogenesis and hypertrophy using small-diameter microtissues generated from 5000 BMSC each. This biology is obfuscated in typical large-diameter pellet cultures, which suffer radial heterogeneity. Here, we investigated if brief stimulation (2 days) of BMSC microtissues with BMP-2 (100 ng/mL) or growth/differentiation factor (GDF-5, 100 ng/mL) was also sufficient to induce chondrogenic differentiation, in a manner comparable to TGF-ß1 (10 ng/mL). Like TGF-ß1, BMP-2 and GDF-5 are reported to stimulate chondrogenic differentiation of BMSCs, but the effects of transient or brief use in culture have not been explored. Hypertrophy is an unwanted outcome in BMSC chondrogenic differentiation that renders engineered tissues unsuitable for use in clinical cartilage repair. Using three BMSC donors, we observed that all GFs facilitated chondrogenesis, although the efficiency and the necessary duration of stimulation differed. Microtissues treated with 2 days or 14 days of TGF-ß1 were both superior at producing extracellular matrix and expression of chondrogenic gene markers compared to BMP-2 and GDF-5 with the same exposure times. Hypertrophic markers increased proportionally with chondrogenic differentiation, suggesting that these processes are intertwined for all three GFs. The rapid action, or "temporal potency", of these GFs to induce BMSC chondrogenesis was found to be as follows: TGF-ß1 > BMP-2 > GDF-5. Whether briefly or continuously supplied in culture, TGF-ß1 was the most potent GF for inducing chondrogenesis in BMSCs.

Knee Osteotomies Can Be Performed Safely In An Ambulatory Setting.

Purpose: The purpose of this study was to assess the rate of hospital admissions, inpatient conversions, reoperations, and complications associated with tibial tubercle osteotomies (TTO), high tibial osteotomies (HTO), and distal femoral osteotomies (DFO) performed at our ambulatory surgery center compared with our inpatient hospital facility. Methods: A retrospective review of patients receiving a TTO, HTO or DFO at our institution between June 2011 and October 2019 was performed. Inclusion criteria consisted of patients undergoing the aforementioned procedures for malalignment, and a minimum of 90-days follow-up. Revision osteotomies, those undergoing an osteotomy for an acute fracture, and those with rule-out criteria for outpatient surgery (ASA > 3, and body mass index >40) were excluded. Complications, including readmission and reoperation, were compared between the two groups using either the Fisher's exact test and independent samples t-test, where applicable, and a P value of <0.05 was considered to be statistically significant. Results: The study included 531 patients undergoing osteotomies (222 ambulatory surgical center [ASC] and 309 hospital) with no patients lost to follow-up in the 90-day postoperative period. No patients operated on at an ASC required transfer to inpatient setting. There were no differences in complication rates, readmission, or reoperation rates among the two groups (4.1% vs 4.9%; P = .8328; 3.1% vs 4.5%, P = .5026; 3.1% vs 4.5%; P = .5026; respectively). Complications, including surgical site infection and arthrofibrosis were not significantly different in the two cohorts, (1.4% vs. 2.6%, P = .341 and 1.4% vs 1%; P = .698, respectively). Conclusions: Osteotomies about the knee performed in an ambulatory setting were safe, with no difference in readmission, reoperation, or postoperative complications compared to those performed at an inpatient hospital. Additionally, no patient required conversion from an outpatient to an inpatient setting. Level of Evidence: Level III, retrospective comparative study.

Direct bone marrow injection of human bone marrow-derived stromal cells into mouse femurs results in greater prostate cancer PC-3 cell proliferation, but not specifically proliferation within the injected femurs.

BACKGROUND: While prostate cancer (PCa) cells most often metastasize to bone in men, species-specific differences between human and mouse bone marrow mean that this pattern is not faithfully replicated in mice. Herein we evaluated the impact of partially humanizing mouse bone marrow with human bone marrow-derived stromal cells (BMSC, also known as "mesenchymal stem cells") on human PCa cell behaviour. METHODS: BMSC are key cellular constituents of marrow. We used intrafemoral injection to transplant 5 × 105 luciferase (Luc) and green fluorescence protein (GFP) expressing human BMSC (hBMSC-Luc/GFP) into the right femur of non-obese diabetic (NOD)-severe combined immunodeficiency (scid) interleukin (IL)-2γ-/- (NSG) mice. Two weeks later, 2.5 × 106 PC-3 prostate cancer cells expressing DsRed (PC-3-DsRed) were delivered into the mice via intracardiac injection. PC-3-DsRed cells were tracked over time using an In Vivo Imaging System (IVIS) live animal imaging system, X-ray and IVIS imaging performed on harvested organs, and PC-3 cell numbers in femurs quantified using flow cytometry and histology. RESULTS: Flow cytometry analysis revealed greater PC-3-DsRed cell numbers within femurs of the mice that received hBMSC-Luc/GFP. However, while there were overall greater PC-3-DsRed cell numbers in these animals, there were not more PC-3-DsRed in the femurs injected with hBMSC-Luc/GFP than in contralateral femurs. A similar proportion of mice in with or without hBMSC-Luc/GFP had bone lessions, but the absolute number of bone lesions was greater in mice that had received hBMSC-Luc/GFP. CONCLUSION: PC-3-DsRed cells preferentially populated bones in mice that had received hBMSC-Luc/GFP, although PC-3-DsRed cells not specifically localize in the bone marrow cavity where hBMSC-Luc/GFP had been transplanted. hBMSC-Luc/GFP appear to modify mouse biology in a manner that supports PC-3-DsRed tumor development, rather than specifically influencing PC-3-DsRed cell homing. This study provides useful insights into the role of humanizing murine bone marrow with hBMSC to study human PCa cell biology.

A Novel Machine Learning Predictive Tool Assessing Outpatient or Inpatient Designation for Medicare Patients Undergoing Total Knee Arthroplasty.

BACKGROUND: Removal of total joint arthroplasty from the inpatient-only list has created significant confusion regarding which patients qualify for an inpatient designation. The purpose of this study is to develop and validate a novel predictive tool for assessing who will be an outpatient vs inpatient after total knee arthroplasty (TKA). METHODS: A cohort of Medicare patients undergoing primary TKA between January 2018 and September 2019 were retrospectively reviewed. Baseline demographics and patient characteristics were obtained, and their distributions for outpatient (less than 2 midnights) and inpatient stay were assessed. Subsequently, a XGBoost machine learning model was trained using 80% of the TKA patients, and the remaining 20% of patients were involved in testing the model's performance in terms of accuracy and the average area under the receive operating characteristic curve. RESULTS: Eight hundred ninety-nine Medicare patients underwent TKA at our institution between January 2018 and September 2019. Of which, 625 patients had outpatients stays, and 274 qualified for inpatient designation. Significant associations were demonstrated between inpatient visits and the following factors: higher body mass index, increased age, better functional scores, multidimensional fatigue inventory, Charlson Comorbidity Index, American Society of Anesthesiologists score, female gender, cardiac history, and the Revised Cardiac Risk Index. The XGBoost model for predicting an inpatient or outpatient stay was 63.3% accurate, with area under the receive operating characteristic curve of 68.8%. CONCLUSIONS: Using readily available key baseline characteristics, functional scores, and comorbidities, this machine-learning model accurately predicts the probability of an "outpatient" vs "inpatient" stay after TKA in the Medicare population. body mass index, age, VR12 functional scores, and multidimensional fatigue inventory scores had the highest influence on this predictive model.

Inhibition of BMP signaling with LDN 193189 can influence bone marrow stromal cell fate but does not prevent hypertrophy during chondrogenesis.

Bone morphogenetic protein (BMP) cascades are upregulated during bone marrow-derived stromal cell (BMSC) chondrogenesis, contributing to hypertrophy and preventing effective BMSC-mediated cartilage repair. Previous work demonstrated that a proprietary BMP inhibitor prevented BMSC hypertrophy, yielding stable cartilage tissue. Because of the significant therapeutic potential of a molecule capable of hypertrophy blockade, we evaluated the capacity of a commercially available BMP type I receptor inhibitor with similar properties, LDN 193189, to prevent BMSC hypertrophy. Using 14-day microtissue chondrogenic induction cultures we found that LDN 193189 permitted BMSC chondrogenesis but did not prevent hypertrophy. LDN 193189 was sufficiently potent to counter mineralization and adipogenesis in response to exogenous BMP-2 in osteogenic induction cultures. LDN 193189 did not modify BMSC behavior in adipogenic induction cultures. Although LDN 193189 is effective in countering BMP signaling in a manner that influences BMSC fate, this blockade is insufficient to prevent hypertrophy.

Collagenase treatment appears to improve cartilage tissue integration but damage to collagen networks is likely permanent.

When repairing cartilage defects a major challenge is achieving high-quality integration between the repair tissue and adjacent native cartilage. Matrix-rich cartilage is not easily remodeled, motivating several studies to trial enzyme treatment of the tissue interface to facilitate remodeling and integration. Studying and optimizing such processes is tedious, as well as potentially expensive, and thus simpler models are needed to evaluate the merits of enzyme treatment on cartilage tissue integration. Herein, we used engineered cartilage microtissues formed from bone marrow-derived stromal cells (BMSC) or expanded articular chondrocytes (ACh) to study the impact of enzyme treatment on cartilage tissue integration and matrix remodeling. A 5-min treatment with collagenase appeared to improve cartilage microtissue integration, while up to 48 h treatment with hyaluronidase did not. Alcian blue and anti-collagen II staining suggested that collagenase treatment did facilitate near seamless integration of cartilage microtissues. Microtissue sections were stained with Picrosirius red and characterized using polarized light microscopy, revealing that individual microtissues contained a collagen network organized in concentric shells. While collagenase treatment appeared to improve tissue integration, assessment of the collagen fibers with polarized light indicated that enzymatically damaged networks were not remodeled nor restored during subsequent culture. This model and these data paradoxically suggest that collagen network disruption is required to improve cartilage tissue integration, but that the disrupted collagen networks are unlikely to subsequently be restored. Future studies should attempt to limit collagen network disruption to the surface of the cartilage, and we recommend using Picrosirius red staining and polarized light to assess the quality of matrix remodeling and integration.

Distal posterolateral corner injury in the setting of multiligament knee injury increases risk of common peroneal palsy.

PURPOSE: The purpose of this study was to identify if the location of posterolateral corner (PLC) injury was predictive of clinical common peroneal nerve (CPN) palsy. METHODS: A retrospective chart review was conducted of patients presenting to our institution with operative PLC injuries. Assessment of concomitant injuries and presence of neurologic injury was completed via chart review and magnetic resonance imaging (MRI) review. A fellowship-trained musculoskeletal radiologist reviewed the PLC injury and categorized it into distal, middle and proximal injuries with or without a biceps femoral avulsion. The CPN was evaluated for signs of displacement or neuritis. RESULTS: Forty-seven operatively managed patients between 2014 and 2019 (mean age-at-injury 29.5 ± 10.7 years) were included in this study. Eleven (23.4%) total patients presented with a clinical CPN palsy. Distal PLC injuries were significantly associated with CPN palsy [9 (81.8%) patients, (P = 0.041)]. Nine of 11 (81.8%) patients with CPN palsy had biceps femoral avulsion (P = 0.041). Of the patients presenting with CPN palsy, only four (36.4%) patients experienced complete neurologic recovery. Three of 7 patients (43%) with an intact CPN had full resolution of their clinically complete CPN palsy at the time of follow-up (482 ± 357 days). All patients presenting with a CPN palsy also had a complete anterior cruciate ligament (ACL) rupture in addition to a PLC injury (P = 0.009), with or without a posterior cruciate ligament (PCL) injury. No patient presenting with an isolated pattern of PCL-PLC injury (those without ACL tears) had a clinical CPN palsy. CONCLUSION: Distal PLC injuries have a strong association with clinical CPN palsy, with suboptimal resolution in the initial post-operative period. Specifically, the presence of a biceps femoris avulsion injury was highly associated with a clinical CPN palsy. Additionally, CPN palsy in the context of PLC injury has a strong association with concomitant ACL injury. Furthermore, the relative rates of involvement of the ACL vs. PCL suggest that specific injury mechanism may have an important role in CPN palsy. LEVEL OF EVIDENCE: IV.

A Novel Machine Learning Predictive Tool Assessing Outpatient or Inpatient Designation for Medicare Patients Undergoing Total Hip Arthroplasty.

BACKGROUND: The Centers for Medicare and Medicaid Services removed total hip arthroplasty (THA) from the inpatient-only list. This has created significant confusion regarding which patients qualify for an inpatient designation. The purpose of this study is to develop and validate a novel predictive tool for preoperatively objectively determining "outpatient" vs "inpatient" status for THA in the Medicare population. METHODS: A cohort of Medicare patients undergoing primary THA between January 2017 and September 2019 was retrospectively reviewed. A machine learning model was trained using 80% of the THA patients, and the remaining 20% was used for testing the model performance in terms of accuracy and the average area under the receiver operating characteristic curve. Feature importance was obtained for each feature used in the model. RESULTS: One thousand ninety-one patients had outpatient stays, and 318 qualified for inpatient designation. Significant associations were demonstrated between inpatient designations and the following: higher BMI, increased patient age, better preoperative functional scores, higher American Society of Anesthesiologist Physical Status Classification, higher Modified Frailty Index, higher Charlson Comorbidity Index, female gender, and numerous comorbidities. The XGBoost model for predicting an inpatient or outpatient stay was 78.7% accurate with the area under the receiver operating characteristic curve to be 81.5%. CONCLUSIONS: Using readily available key baseline characteristics, functional scores and comorbidities, this machine-learning model accurately predicts an "outpatient" or "inpatient" stay after THA in the Medicare population. BMI, age, functional scores, and American Society of Anesthesiologist Physical Status Classification had the highest influence on this predictive model.

The Association Between Clopidogrel and Gastrointestinal Bleeding After Primary Total Joint Arthroplasty.

BACKGROUND: Anticoagulation after total joint arthroplasty has been demonstrated to reduce venous thromboembolism. However, anticoagulation can lead to adverse bleeding events. The purpose of this study was to assess if an association exists between specific anticoagulation modalities, such as clopidogrel, and postoperative gastrointestinal (GI) bleeding. METHODS: A prospective cohort of Medicare patients undergoing total joint arthroplasty from 2017 to 2019 (3535 patients) was retrospectively reviewed. The baseline characteristics and anticoagulation methods were compared between the "GI bleed" cohort and the "non-GI bleed cohort." Independent t-tests were conducted for continuous variables, while chi-squared analysis was conducted for dichotomous variables. RESULTS: Thirteen patients (0.42%) sustained a postoperative complication of a GI bleed. The mean age for patients sustaining a GI bleed was 69.23 years compared with 72.30 years for the non-GI bleed cohort (P = .11). Six patients who sustained a GI bleed (46%) were on an anticoagulation therapy other than aspirin, and this trended toward significance (P = .09). Five patients who sustained a GI bleed (38%) were on clopidogrel (P < .01). Seven percent of patients on clopidogrel sustained a postoperative GI bleed (P < .01). None of the patients who sustained a postoperative GI bleed had a history of peptic ulcer disease. CONCLUSION: Patients on clopidogrel in the acute perioperative period demonstrated a strong association with the complication of postoperative GI bleeding. Arthroplasty surgeons should be aware of this association to educate and monitor patients on clopidogrel therapy and to work as part of interdisciplinary teams to assess the risks vs benefits of perioperative clopidogrel.

Human bone marrow-derived stromal cell behavior when injected directly into the bone marrow of NOD-scid-gamma mice pre-conditioned with sub-lethal irradiation.

BACKGROUND: Direct bone marrow injection of cells into murine marrow cavities is used in a range of cell characterization assays and to develop disease models. While human bone marrow-derived stromal cells (hBMSC, also known as mesenchymal stem cells (MSC)) are frequently described in therapeutic applications, or disease modeling, their behavior following direct injection into murine bone marrow is poorly characterized. Herein, we characterized hBMSC engraftment and persistence within the bone marrow of NOD-scid interleukin (IL)-2γ-/- (NSG) mice with or without prior 2 Gy total-body γ-irradiation of recipient mice. METHODS: One day after conditioning NSG mice with sublethal irradiation, 5 × 105 luciferase (Luc) and green fluorescent protein (GFP)-expressing hBMSC (hBMSC-Luc/GFP) were injected into the right femurs of animals. hBMSC-Luc/GFP were tracked in live animals using IVIS imaging, and histology was used to further characterize hBMSC location and behavior in tissues. RESULTS: hBMSC-Luc/GFP number within injected marrow cavities declined rapidly over 4 weeks, but prior irradiation of animals delayed this decline. At 4 weeks, hBMSC-Luc/GFP colonized injected marrow cavities and distal marrow cavities at rates of 2.5 ± 2.2% and 1.7 ± 1.9% of total marrow nucleated cells, respectively in both irradiated and non-irradiated mice. In distal marrow cavities,  hBMSC were not uniformly distributed and appeared to be co-localized in clusters, with the majority found in the endosteal region. CONCLUSIONS: While significant numbers of hBMSC-Luc/GFP could be deposited into the mouse bone marrow via direct bone marrow injection, IVIS imaging indicated that the number of hBMSC-Luc/GFP in that bone marrow cavity declined with time. Irradiation of mice prior to transplant only delayed the rate of hBMSC-Luc/GFP population decline in injected femurs. Clusters of hBMSC-Luc/GFP were observed in the histology of distal marrow cavities, suggesting that some transplanted cells actively homed to distal marrow cavities. Individual cell clusters may have arisen from discrete clones that homed to the marrow, and then underwent modest proliferation. The transient high-density population of hBMSC within the injected femur, or the longer-term low-density population of hBMSC in distal marrow cavities, offers useful models for studying disease or regenerative processes. Experimental designs should consider how relative hBMSC distribution and local hBMSC densities evolve over time.