Beyond SEP-1 Compliance: Assessing the Impact of Antibiotic Overtreatment and Fluid Overload in Suspected Septic Patients.

BACKGROUND: The Centers for Medicare and Medicaid Services (CMS) developed the Severe Sepsis and Septic Shock Performance Measure bundle (SEP-1) metric to improve sepsis care, but evidence supporting this bundle is limited and harms secondary to compliance have not been investigated. OBJECTIVE: This study investigates the effect of an emergency department (ED) sepsis quality-improvement (QI) effort to improve CMS SEP-1 compliance, looking specifically at antibiotic overtreatment and harm from fluid resuscitation. METHODS: This was a retrospective observational study conducted between March and July 2021 with patients for whom a sepsis order set was initiated. The primary outcomes included the number of patients treated with antibiotics who were ultimately deemed nonseptic and the number of patients who developed pulmonary edema, with or without need for positive pressure ventilation (PPV), within 48 h of receiving a 30 mL/kg fluid bolus. Data were collected via nonblinded chart reviews, with a free marginal κ-calculation indicating excellent interrater reliability. RESULTS: The study cohort included 273 patients, 170 (62.3%) who were ultimately determined to be septic and 103 (37.7%) who were nonseptic. Of the 103 nonseptic patients, 82 (79.6%) received antibiotics in the ED. Of the 121 patients (44.3%) who received a 30 mL/kg bolus, 5 patients (4.1%) developed pulmonary edema and 0 of 121 patients required PPV within 48 h. CONCLUSIONS: The QI effort led to moderate rates of antibiotic overtreatment and very few patients developed pulmonary edema due to a 30 mL/kg fluid bolus.

Perceived Value of Osteopathic Recognition.

BACKGROUND AND OBJECTIVES: In 2020 the Accreditation Council on Graduate Medical Education (ACGME) became the sole accrediting body for osteopathic and allopathic residency programs, with an option for programs to apply for Osteopathic Recognition (OR) to distinguish their training in osteopathic principles and practice. There is limited research regarding this transition. The goal of our study was to assess the perceived value of OR and perceived difficulty of obtaining OR for family medicine residency programs. METHODS: We performed analyses regarding the difficulty of obtaining OR status and the value of OR and Osteopathic Principles and Practice (OPP) using questions on the 2020 Council of Academic Family Medicine Educational Research Alliance (CERA) omnibus survey. RESULTS: Of the 280 program directors (PD) responding, 69 (24.6%) had OR status, 126 (45.0%) were considering applying or would apply if needed resources were available, and 85 (30.4%) were not considering OR. Of the 73 PDs reporting on experience with the OR process, 28 (38.4%) found it "very smooth," 30 (41.1%) found it "a little bumpy," and 15 (20.5%) found it "very bumpy"; 87.0% of PDs (60 of 69) with OR felt it had value in recruiting DO students and 31.8% (22/69) in recruiting MD students; 86.9% of programs with OR status perceived OPP to be somewhat or very valuable in enhancing patient satisfaction compared to 77% of those considering OR and 44.7% not considering OR. CONCLUSIONS: Program directors perceive value in OR status for recruiting and in osteopathic practice for patient care. Since 75.4% of responding program directors have or are interested in achieving OR status, further research is needed on its benefits and barriers.

Hemodynamic profile effects of PM101 amiodarone formulation in patients with post-operative tachyarrhythmias.

Amiodarone may be considered for patients with junctional ectopic tachycardia refractory to treatment with sedation, analgesia, cooling, and electrolyte replacements. There are currently no published pediatric data regarding the hemodynamic effects of the newer amiodarone formulation, PM101, devoid of hypotensive agents used in the original amiodarone formulation. We performed a single-center, retrospective, descriptive study from January 2012 to December 2020 in a pediatric ICU. Thirty-three patients were included (22 male and 11 female) between the ages of 1.1 and 1,460 days who developed post-operative junctional ectopic tachycardia or other tachyarrhythmias requiring PM101. Data analysis was performed on hemodynamic parameters (mean arterial pressures and heart rate) and total PM101 (mg/kg) from hour 0 of amiodarone administration to hour 72. Adverse outcomes were defined as Vasoactive-Inotropic Score >20, patients requiring ECMO or CPR, or patient death. There was no statistically significant decrease in mean arterial pressures within the 6 hours of PM101 administration (p > 0.05), but there was a statistically significant therapeutic decrease in heart rate for resolution of tachyarrhythmia (p < 0.05). Patients received up to 25 mg/kg in an 8-hour time for rate control. Average rate control was achieved within 11.91 hours and average rhythm control within 62 hours. There were four adverse events around the time of PM101 administration, with three determined to not be associated with the medication. PM101 is safe and effective in the pediatric cardiac surgical population. Our study demonstrated that PM101 can be used in a more aggressive dosing regimen than previously reported in pediatric literature with the prior formulation.

Transseptal puncture during catheter ablation associated with higher radiation exposure.

BACKGROUND: Electroanatomic mapping systems are increasingly used during ablations to decrease the need for fluoroscopy and therefore radiation exposure. For left-sided arrhythmias, transseptal puncture is a common procedure performed to gain access to the left side of the heart. We aimed to demonstrate the radiation exposure associated with transseptal puncture. METHODS: Data were retrospectively collected from the Catheter Ablation with Reduction or Elimination of Fluoroscopy registry. Patients with left-sided accessory pathway-mediated tachycardia, with a structurally normal heart, who had a transseptal puncture, and were under 22 years of age were included. Those with previous ablations, concurrent diagnostic or interventional catheterisation, and missing data for fluoroscopy use or procedural outcomes were excluded. Patients with a patent foramen ovale who did not have a transseptal puncture were selected as the control group using the same criteria. Procedural outcomes were compared between the two groups. RESULTS: There were 284 patients in the transseptal puncture group and 70 in the patent foramen ovale group. The transseptal puncture group had a significantly higher mean procedure time (158.8 versus 131.4 minutes, p = 0.002), rate of fluoroscopy use (38% versus 7%, p < 0.001), and mean fluoroscopy time (2.4 versus 0.6 minutes, p < 0.001). The acute success and complication rates were similar. CONCLUSIONS: Performing transseptal puncture remains a common reason to utilise fluoroscopy in the era of non-fluoroscopic ablation. Better tools are needed to make non-fluoroscopic transseptal puncture more feasible.

Role of Prophylactic Sartorius Flaps for Preventing Deep Space Infection in Lower Extremity Revascularization.

Groin infections in vascular surgery are common and compromise the goal of limb preservation. Strategies to prevent deep space infection (DSI) include incision orientation and muscle flaps. Literature evaluating prophylactic flaps preventing DSI is scarce. We aimed to compare prophylactic sartorius flaps to layered closure in preventing readmission for DSI, along with the effect of incision orientation. This was a retrospective study of vascular surgery patients at a single institution with femoral artery exposure from 2017-2021. Patients with active groin infections were excluded. Prophylactic sartorius flaps were compared to those with layered closure regarding 30-day hospital readmission for DSI. Oblique versus vertical incisions was compared regarding the primary outcome. Fifty-three patients received sartorius flaps, and 122 received layered closure. Seventy patients had oblique incisions, and 105 patients had vertical incisions. Sartorius flaps had a higher rate of previous groin surgery compared to layered closure (45.3% vs. 24.7%, p<0.01). Vertical incisions had a higher rate of previous groin surgery (38.1% vs. 20.0%, p<0.02), while oblique incisions had a higher rate of obesity (24.3% vs. 8.6%, p<0.01). There was a lower rate of DSI in sartorius flaps compared to layered closure (1.9% vs. 6.6%, p=2.80), although not statistically significant due to lack of power. There was no difference in DSI in the oblique versus vertical incisions (4.3% and 5.7%, p=0.760). Patients with prophylactic sartorius flaps experienced fewer DSI, although further evaluation with increased sample size is required for adequate study power. We believe sartorius flaps are a simple solution to prevent groin complications.

Long-term treatment retention of an emergency department initiated medication for opioid use disorder program.

INTRODUCTION: Medication for Opioid Use Disorder (MOUD) has been shown to decrease mortality, reduce overdoses, and increase treatment retention for patients with opioid use disorder (OUD) and has become the state-of-the-art treatment strategy in the emergency department (ED). There is little evidence on long-term (6 and 12 month) treatment retention outcomes for patients enrolled in MOUD from the ED. METHODS: A prospective observational study used a convenience sample of patients seen at one community hospital ED over 12 months. Patients >18 years with OUD were eligible for MOUD enrollment. After medical screening, patients were evaluated by the addiction care coordinator (ACC) who evaluated and counselled the patient and if eligible, directly connected them with an addiction medicine appointment. Once enrolled, the patient received treatment with buprenorphine in the ED. A chart review was completed for all enrollments during the first year of the program. Treatment retention was determined by review of the prescription drug monitoring program and defined as patients receiving regular suboxone prescriptions at 6 and 12 months after index ED visit date. RESULTS: From June 2018 - May 2019 the ACCs evaluated patients during 691 visits, screening 571 unique patients. Of the 571 unique patients screened, 279 (48.9%) were enrolled into the MOUD program. 210 (75.3%) attended their first addiction medicine appointment, 151 (54.1%) were engaged in treatment at 1 month, 120 (43.0%) at 3 months, 105 (37.6%) at 6 months, and 97 (34.8%) at 12 months post index ED visit. Self-pay insurance status was associated with a significantly decrease in the odds of long-term treatment retention. CONCLUSION: Our ED-initiated MOUD program, in partnership with local addiction medicine services, produced high rates of long-term treatment retention.

Safety of Inpatient Dofetilide Initiation per Cardiology Services: A Retrospective Review.

INTRODUCTION: Dofetilide is an antiarrhythmic medication that has the potential to cause life threatening arrhythmias, such as Torsade de pointes. The 2014 ACC/AHA/HRS guidelines for the management of patients with atrial fibrillation state that patients need to meet certain criteria to be initiated on dofetilide. Patients who are not initiated on this therapy according to the guideline criteria are likely to be at higher risk of adverse reactions. METHODS: This is a single center, retrospective chart review of patients who were initiated on dofetilide from July 2016-December 2019. Patients included in the study were initiated on dofetilide as a new antiarrhythmic and monitored inpatient for 3 days. The primary outcome was a composite of incidence of cardiac arrhythmias, cardiac arrest, cardiac death, and cardiac related hospital readmission. RESULTS: There were 224 patients included in the analysis: 190 patients who were initiated on dofetilide inappropriately and 34 that were initiated appropriately. The primary outcome (composite of cardiac arrhythmia, cardiac arrest, cardiac death, and hospital readmission) was statistically significant with more patients experiencing an outcome in the group initiated inappropriately. CONCLUSIONS: Patients are placed at a higher risk of adverse reactions when this potentially dangerous antiarrhythmic medication is not used according to the protocol set forth by the guidelines. Practitioners should use caution when prescribing dofetilide. Other antiarrhythmic medications or non-pharmacologic options should be considered due to the incidence of these dangerous adverse reactions.

Simulation-based Training Curriculum for the Management of Vaginal Cuff Dehiscence and Evisceration.

Objective Vaginal cuff dehiscence with evisceration (VCDE) is a serious, life-threatening complication of hysterectomy. Due to the high volume of hysterectomies performed in the U.S each year, it is likely that a practitioner will encounter VCDE during their career. Due to its infrequent occurrence, residents receive little exposure to it during training. Delayed diagnosis of VCDE can impede proper management and lead to severe, long-term complications or death. Our goal was to provide an opportunity for resident physicians to identify VCDE and practice performing a reduction of prolapsed bowel and vaginal cuff repair through hands-on simulation in hopes that the simulation would improve the residents' confidence and knowledge in recognizing and managing future VCDE cases. Methods Obstetrics and Gynecology residents postgraduate year (PGY) 1-4 participated in this study (n=13). Before and after the simulated case, a knowledge test covering VCDE recognition and management and a confidence survey were given to the participants. A gynecologic mannequin was modified by placing simulated bowel into the abdominal cavity with a portion extending through a vaginal cuff and protruding from the vaginal introitus. For the simulation, a hemodynamically unstable patient presented with findings consistent with a VCDE. Once the decision to proceed to surgery was made, participants were transferred to a simulated operating room where they performed a reduction of prolapsed bowel and vaginal cuff closure either laparoscopically or vaginally on the mannequin. A debriefing session was held post-simulation to discuss management and thought processes, as well as reflect on their performance and discuss improvement strategies for future cases. Finally, the residents participated in a brief didactic lecture on education about the incidence, presentation, and management of VCDE. Results Analysis of the knowledge questionnaires showed the median score and interquartile range (IQR) pre- and post-simulation was 15(12-28) and 20(19-22) respectively, with a median score increase (and IQR) of 5(3.5-8.5) (p=0.001). The confidence score had pre- and post-simulation median scores (and IQRs) of 28(20-34.5) and 40(37.5-46) respectively, with a median score increase (and IQR) of 15(8-20.5) (p=0.001). Conclusions Our intervention improved residents' knowledge and confidence in recognizing VCDE, identifying the need for surgical management, and performing a reduction of prolapsed bowel and vaginal cuff repair.

Prehospital Blood Glucose Testing as a Predictor of Impending Hypotension in Adult Trauma Patients.

OBJECTIVE: Stress-induced hyperglycemia has been found to increase hemorrhagic shock, morbidity, and mortality in the trauma patient. The purpose of this study is to evaluate whether prehospital point-of-care glucose is an independent predictor of hypotension in the adult trauma patient transported by air ambulance to the receiving trauma center. METHODS: This retrospective chart review evaluated adult, nondiabetic trauma patients transported by air ambulance at 3 programs in the Midwest for the calendar year 2018. A total of 107 patients met the inclusion criteria. The primary analysis was the determination of an optimal cutoff for the blood glucose diagnostic for predicting a hypotensive outcome followed by chi-square incidence comparison. RESULTS: The optimal diagnostic cutoff point using Youden's index (J) was determined to be a blood glucose value of 220 mg/dL or greater. Initial glucose values were associated with an increased relative risk of a hypotension outcome (P = .040). Glucose dichotomy was also associated with a mean decrease in systolic blood pressure during transport (P = .016). CONCLUSION: The findings in this study indicate a point-of-care glucose measurement greater than 220 mg/dL should prompt prehospital clinicians to initiate aggressive balanced resuscitation before arrival at the receiving trauma center in order to prevent worsening hypotension and hemorrhagic shock.

Simulation-based Training in Ectopic Pregnancy and Salpingostomy.

Objective Ectopic pregnancy leads to approximately 3% of deaths in pregnancy. Surgical management is indicated when patients are hemodynamically unstable or have signs of a ruptured ectopic pregnancy. Salpingectomy is more commonly performed, but salpingostomy is preferred in a patient with prior salpingectomy with a desire for future pregnancy. Due to the lack of exposure, salpingostomy is not frequently performed and most residents do not feel adequately trained. Our goal was to provide a hands-on simulation about ectopic pregnancy and salpingostomy in hopes that the simulation will improve the resident's confidence and knowledge in recognizing an ectopic pregnancy, identifying an appropriate candidate for surgical management, and performing a salpingostomy. Methods The educational initiative was aimed towards postgraduate year (PGY) 1-4 OB/GYN residents (n=11). Knowledge and confidence questionnaires were given to participants prior to and post-simulation. A gynecologic mannequin was modified by taking the existing pelvic organs and creating a tubal pregnancy. In the first part of the simulation, a hemodynamically unstable patient presented with lab and imaging findings consistent with an ectopic pregnancy. Once recognized and the decision made for surgical intervention, participants were transferred to a simulated operating room where they performed salpingostomy or salpingectomy on the mannequin. The simulation was followed by a debriefing session to discuss the actions and thought processes of participants, provide reflection, and incorporate improvement opportunities for future cases. Finally, participants engaged in a didactic lecture where they were educated about the incidence, presentation, and management of tubal ectopic pregnancy. Results Analysis of the knowledge questionnaires showed the median score pre- and post-intervention was 9 and 12, respectively, with a median change of 3 (p=0.001). The median confidence value pre- and post-intervention were 28 and 42, respectively, with a median value change of 12 (p<0.001). Conclusion Our intervention improved residents' confidence and knowledge in recognizing an ectopic pregnancy, identifying an appropriate candidate for surgical management, and performing a salpingostomy.

Emergency Undocking Curriculum in Robotic Surgery.

Introduction Traditional instruction for robotic surgery is typically devoid of training that addresses the delineation of interprofessional roles for operating room personnel. An emergency undocking scenario was developed for robotic surgeons with the objectives of improving time to access the patient, provider knowledge of and confidence in emergency undocking, completion of predetermined critical actions, and delineation of operating room personnel roles. Methods Over one month, participants joined in three sessions: Session 1 - formative, Session 2 - review, and Session 3 - summative. Embedded standardized participants (ESPs) represented members of the interprofessional team. Prior to entering the operating room for Sessions 1 and 3, trainees were asked to complete a confidence survey and multiple choice questionnaire (MCQ) for knowledge assessment. Participants were randomized to one of two cases and participated in the reciprocal case for the final session four weeks later. Following Session 1, participants underwent an educational intervention, including the proper technique for emergency undocking, emphasis on operating room personnel roles, and hands-on practice. Obstetrics and Gynecology (OBGYN) residents in post-graduate Years 2-4 and attending physicians with robotics privileges at Summa Health Akron Campus or Cleveland Clinic Akron General Medical Center were invited to participate. A total of 21 participants enrolled and finished the study. Results Among the 21 participants, there was a significant increase in the baseline level of knowledge (p-value=0.001) and in the confidence of surgeons when faced with an emergency undocking after the completion of our curriculum (p-value=0.003). Additionally, an improvement in the undocking times (p-value<0.001) and an increase in the critical actions performed (p-value=0.002) were observed. Conclusion The results of this study demonstrate that incorporating this curriculum into the training programs of robotic surgeons is an effective way to improve the surgical skill of emergency undocking.

Type 2 Diabetes Prescribing Habits amongst Providers to an Underserved Population.

INTRODUCTION: Type 2 diabetes mellitus (T2DM) affected over 30 million individuals in the United States as of 2015. Due to the national diabetes guidelines recommending drug selection based on several patient specific factors and varying formulary restrictions, prescribers are often inundated when selecting treatment. Currently, limited evidence is available regarding the primary factors influencing prescribers' drug therapy selection. OBJECTIVES: The purpose of this study was to identify factors that influence providers during T2DM medication selection. METHODS: The study was conducted with providers at a large, academic, safety net health system. All prescribers were sent an electronic, optional and anonymous survey. Prescribers treating T2DM in non-pregnant adult patients were the only prescribers assessed. Factors evaluated were: cost, A1c, comorbidities, adherence, weight, tolerability, patient limitations, and use of guidelines. RESULTS: A total of 86 prescribers responded, yielding a response rate of 31%. The respondents included physicians (56.3%), nurse practitioners (21.8%), medical residents (18.4%), and fellows (3.4%); with the majority practicing in internal or family medicine (47.1%). The most frequently prescribed T2DM medications included: metformin (83.8%), insulin (78.1%), and sulfonylureas (64.8%). Cost and A1c elevation were two of the major factors influencing prescribing of metformin (94.1% and 81.2%), insulin (57.4% and 69.6%), and sulfonylureas (81.2% and 89.9%) respectively. Due to cost concerns, respondents reported rarely or never prescribing glucagon-like peptide-1 agonists (GLP-1RA) and sodium-glucose co-transporter-2 inhibitors (SGLT2i) despite recognizing benefits on diabetes related comorbidities. CONCLUSION: Although current literature from the national guidelines encourages the use of GLP-1RA and SGLT2i as first-line options after metformin in T2DM, these classes of medications were not reported among the most commonly prescribed despite providers correctly identifying positive medication attributes such as cardio- and nephroprotection and weight loss. However, cost of these medications appears to outweigh the benefits when selecting medication therapy.

Platelet rich plasma for photodamaged skin: A pilot study.

INTRODUCTION: Despite increasing popularity of platelet-rich plasma (PRP) in treating aging facial skin, the quality of evidence supporting its use is poor due to the lack of consistent methods of its preparation and application. OBJECTIVE: This study was conducted to assess treatment efficacy and patient satisfaction with a single PRP treatment prepared with a simplified preparation and application technique. METHODS: Four millilitre of PRP were injected into 6 standardized points on each side of the face. Outcomes were assessed by independent physician evaluation of pretreatment and posttreatment photographs using the Wrinkle Severity Rating Scale (WSRS) and Global Aesthetic Improvement Scale (GAIS). In addition, patient-reported outcomes were evaluated using the FACE-Q. RESULTS: Thirty-one participants ranging from 27 to 71 years of age (median, 38; IQR 32-58) were recruited for this study. Posttreatment WSRS scores improved in only 1 patient; the GAIS scores of 14 patients indicated aesthetic improvement. Analysis of FACE-Q scores revealed statistically significant increases in participant satisfaction with overall facial appearance and cheeks. The most frequently reported adverse effects were tenderness (23.4%; 7 of 31), facial tightness (20.0%; 6 of 31), and swelling (20.0%; 6 of 31). CONCLUSIONS: A simple method of PRP preparation offers modest benefit in treating the effects of skin aging and photodamage. Future research studies should alter our methods using a stepwise approach to optimize the treatment of aging facial skin with PRP.

Impact of Pharmacist-Led Diabetes Management in Primary Care Clinics.

PURPOSE: Current literature supports pharmacists effectively lower hemoglobin A1c (HbA1c) in diabetic patients. Little data exists on pharmacists' effects on comorbidity management, patient satisfaction, or the financial viability of these positions. This study looked to assess the impact of pharmacists on diabetes management compared to usual care. METHODS: This multi-site, two-part study includes a retrospective chart review of patients referred to the pharmacist versus usual care within a large academic health system. The pharmacists collaborated under a consult agreement with primary care physicians. The second part of the study assessed patient satisfaction through an abbreviated CG-CAHPS survey. RESULTS: A total of 206 patients with diabetes for an average of 12 years were included. The average patient age was 62 years with 60% of patients identifying as female and 81% as African-American. Patients were enrolled in a 2:1 fashion with 138 patients in the pharmacist-management group. Average baseline HbA1c was 10.1% in the pharmacist-management group and 9.3% in the usual care group (p= 0.0125). At 6 months, the mean change in HbA1c was -2.17% and 0.48% for the intervention and control groups respectively (p < 0.001). CONCLUSION: Pharmacists are effective at lowering HbA1c in primary care clinics, and patients were highly satisfied with these services. While direct revenue from this service did not meet cost, the pharmacist did positively affect outcomes that contribute to reimbursement.

In Vivo Assessment of Bone Regeneration in Alginate/Bone ECM Hydrogels with Incorporated Skeletal Stem Cells and Single Growth Factors.

The current study has investigated the use of decellularised, demineralised bone extracellular matrix (ECM) hydrogel constructs for in vivo tissue mineralisation and bone formation. Stro-1-enriched human bone marrow stromal cells were incorporated together with select growth factors including VEGF, TGF-ß3, BMP-2, PTHrP and VitD3, to augment bone formation, and mixed with alginate for structural support. Growth factors were delivered through fast (non-osteogenic factors) and slow (osteogenic factors) release PLGA microparticles. Constructs of 5 mm length were implanted in vivo for 28 days within mice. Dense tissue assessed by micro-CT correlated with histologically assessed mineralised bone formation in all constructs. Exogenous growth factor addition did not enhance bone formation further compared to alginate/bone ECM (ALG/ECM) hydrogels alone. UV irradiation reduced bone formation through degradation of intrinsic growth factors within the bone ECM component and possibly also ECM cross-linking. BMP-2 and VitD3 rescued osteogenic induction. ALG/ECM hydrogels appeared highly osteoinductive and delivery of angiogenic or chondrogenic growth factors led to altered bone formation. All constructs demonstrated extensive host tissue invasion and vascularisation aiding integration and implant longevity. The proposed hydrogel system functioned without the need for growth factor incorporation or an exogenous inducible cell source. Optimal growth factor concentrations and spatiotemporal release profiles require further assessment, as the bone ECM component may suffer batch variability between donor materials. In summary, ALG/ECM hydrogels provide a versatile biomaterial scaffold for utilisation within regenerative medicine which may be tailored, ultimately, to form the tissue of choice through incorporation of select growth factors.

Regionally-derived cell populations and skeletal stem cells from human foetal femora exhibit specific osteochondral and multi-lineage differentiation capacity in vitro and ex vivo.

BACKGROUND: Adult skeletal stem cells (SSCs) often exhibit limited in vitro expansion with undesirable phenotypic changes and loss of differentiation capacity. Foetal tissues offer an alternative cell source, providing SSCs which exhibit desirable differentiation capacity over prolonged periods, ideal for extensive in vitro and ex vivo investigation of fundamental bone biology and skeletal development. METHODS: We have examined the derivation of distinct cell populations from human foetal femora. Regionally isolated populations including epiphyseal and diaphyseal cells were carefully dissected. Expression of the SSC marker Stro-1 was also found in human foetal femora over a range of developmental stages and subsequently utilised for immuno-selection. RESULTS: Regional populations exhibited chondrogenic (epiphyseal) and osteogenic (diaphyseal) phenotypes following in vitro and ex vivo characterisation and molecular analysis, indicative of native SSC maturation during skeletal development. However, each population exhibited potential for induced multi-lineage differentiation towards bone (bone nodule formation), cartilage (proteoglycan and mucopolysaccharide deposition) and fat (lipid deposition), suggesting the presence of a shared stem cell sub-population. This shared sub-population may be comprised of Stro-1+ cells, which were later identified and immuno-selected from whole foetal femora exhibiting multi-lineage differentiation capacity in vitro and ex vivo. CONCLUSIONS: Distinct populations were isolated from human foetal femora expressing osteochondral differentiation capacity. Stro-1 immuno-selected SSCs were isolated from whole femora expressing desirable multi-lineage differentiation capacity over prolonged in vitro expansion, superior to their adult-derived counterparts, providing a valuable cell source with which to study bone biology and skeletal development.

Prospective isolation of human bone marrow stromal cell subsets: A comparative study between Stro-1-, CD146- and CD105-enriched populations.

Stro-1 has proved an efficacious marker for enrichment of skeletal stem and progenitor cells although isolated populations remain heterogeneous, exhibiting variable colony-forming efficiency and osteogenic differentiation potential. The emerging findings that skeletal stem cells originate from adventitial reticular cells have brought two further markers to the fore including CD146 and CD105 (both primarily endothelial and perivascular). This study has compared CD146-, CD105- and Stro-1 (individual and in combination)-enriched human bone marrow stromal cell subsets and assessed whether these endothelial/perivascular markers offer further selection over conventional Stro-1. Fluorescent cell sorting quantification showed that CD146 and CD105 both targeted smaller (2.22% ± 0.59% and 6.94% ± 1.34%, respectively) and potentially different human bone marrow stromal cell fractions compared to Stro-1 (16.29% ± 0.78%). CD146+, but not CD105+, cells exhibited similar alkaline phosphatase-positive colony-forming efficiency in vitro and collagen/proteoglycan deposition in vivo to Stro-1+ cells. Molecular analysis of a number of select osteogenic and potential osteo-predictive genes including ALP, CADM1, CLEC3B, DCN, LOXL4, OPN, POSTN and SATB2 showed Stro-1+ and CD146+ populations possessed similar expression profiles. A discrete human bone marrow stromal cell fraction (2.04% ± 0.41%) exhibited positive immuno-labelling for both Stro-1 and CD146. The data presented here show that CD146+ populations are comparable but not superior to Stro-1+ populations. However, this study demonstrates the critical need for new candidate markers with which to isolate homogeneous skeletal stem cell populations or skeletal stem cell populations which exhibit homogeneous in vitro/in vivo characteristics, for implementation within tissue engineering and regenerative medicine strategies.

In search of the skeletal stem cell: isolation and separation strategies at the macro/micro scale for skeletal regeneration.

Skeletal stem cells (SSCs) show great capacity for bone and cartilage repair however, current in vitro cultures are heterogeneous displaying a hierarchy of differentiation potential. SSCs represent the diminutive true multipotent stem cell fraction of bone marrow mononuclear cell (BMMNC) populations. Endeavours to isolate SSCs have generated a multitude of separation methodologies. SSCs were first identified and isolated by their ability to adhere to culture plastic. Once isolated, further separation is achieved via culture in selective or conditioned media (CM). Indeed, preferential SSC growth has been demonstrated through selective in vitro culture conditions. Other approaches have utilised cell morphology (size and shape) as selection criteria. Studies have also targeted SSCs based on their preferential adhesion to specified compounds, individually or in combination, on both macro and microscale platforms. Nevertheless, most of these methods which represent macroscale function with relatively high throughput, yield insufficient purity. Consequently, research has sought to downsize isolation methodologies to the microscale for single cell analysis. The central approach is identification of the requisite cell populations of SSC-specific surface markers that can be targeted for isolation by either positive or negative selection. SELEX and phage display technology provide apt means to sift through substantial numbers of candidate markers. In contrast, single cell analysis is the paramount advantage of microfluidics, a relatively new field for cell biology. Here cells can be separated under continuous or discontinuous flow according to intrinsic phenotypic and physicochemical properties. The combination of macroscale quantity with microscale specificity to generate robust high-throughput (HT) technology for pure SSC sorting, isolation and enrichment offers significant implications therein for skeletal regenerative strategies as a consequence of lab on chip derived methodology.

Engineering embryonic stem-cell aggregation allows an enhanced osteogenic differentiation in vitro.

Pluripotent embryonic stem (ES) cells hold great promise for the field of tissue engineering, with numerous studies investigating differentiation into various cell types including cardiomyocytes, chondrocytes, and osteoblasts. Previous studies have detailed osteogenic differentiation via dissociated embryoid body (EB) culture in osteoinductive media comprising of ascorbic acid, beta-glycerophosphate, and dexamethasone. It is hoped that these osteogenic cultures will have clinical application in bone tissue repair and regeneration and pharmacological testing. However, differentiation remains highly inefficient and generates heterogeneous populations. We have previously reported an engineered three-dimensional culture system for controlled ES cell-ES cell interaction via the avidin-biotin binding complex. Here we investigate the effect of such engineering on ES cell differentiation. Engineered EBs exhibit enhanced osteogenic differentiation assessed by cadherin-11, Runx2, and osteopontin expression, alkaline phosphatase activity, and bone nodule formation. Results show that cultures produced from intact EBs aggregated for 3 days generated the greatest levels of osteogenic differentiation when cultured in osteoinductive media. However, when cultured in control media, only engineered samples appeared to exhibit bone nodule formation. In addition, polymerase chain reaction analysis revealed a decrease in endoderm and ectoderm expression within engineered samples. This suggests that engineered ES cell aggregation has increased mesoderm homogeneity, contributing to enhanced osteogenic differentiation.

Controlled embryoid body formation via surface modification and avidin-biotin cross-linking.

Cell-cell interaction is an integral part of embryoid body (EB) formation controlling 3D aggregation. Manipulation of embryonic stem (ES) cell interactions could provide control over EB formation. Studies have shown a direct relationship between EB formation and ES cell differentiation. We have previously described a cell surface modification and cross-linking method for influencing cell-cell interaction and formation of multicellular constructs. Here we show further characterisation of this engineered aggregation. We demonstrate that engineering accelerates ES cell aggregation, forming larger, denser and more stable EBs than control samples, with no significant decrease in constituent ES cell viability. However, extended culture >/=5 days reveals significant core necrosis creating a layered EB structure. Accelerated aggregation through engineering circumvents this problem as EB formation time is reduced. We conclude that the proposed engineering method influences initial ES cell-ES cell interactions and EB formation. This methodology could be employed to further our understanding of intrinsic EB properties and their effect on ES cell differentiation.