Iliac Crest Bone Marrow Aspirates Research Articles

Functionality of bone marrow mesenchymal stromal cells derived from head and neck cancer patients – A FDA-IND enabling study regarding MSC-based treatments for radiation-induced xerostomia

PurposeSalivary dysfunction is a significant side effect of radiation therapy for head and neck cancer (HNC). Preliminary data suggests that mesenchymal stromal cells (MSCs) can improve salivary function. Whether MSCs from HNC patients who have completed chemoradiation are functionally similar to those from healthy patients is unknown. We performed a pilot clinical study to determine whether bone marrow-derived MSCs [MSC(M)] from HNC patients could be used for the treatment of RT-induced salivary dysfunction. MethodsAn IRB-approved pilot clinical study was undertaken on HNC patients with xerostomia who had completed treatment two or more years prior. Patients underwent iliac crest bone marrow aspirate and MSC(M) were isolated and cultured. Culture-expanded MSC(M) were stimulated with IFNγ and cryopreserved prior to reanimation and profiling for functional markers by flow cytometry and ELISA. MSC(M) were additionally injected into mice with radiation-induced xerostomia and the changes in salivary gland histology and salivary production were examined. ResultsA total of six subjects were enrolled. MSC(M) from all subjects were culture expanded to > 20 million cells in a median of 15.5 days (range 8–20 days). Flow cytometry confirmed that cultured cells from HNC patients were MSC(M). Functional flow cytometry demonstrated that these IFNγ-stimulated MSC(M) acquired an immunosuppressive phenotype. IFNγ-stimulated MSC(M) from HNC patients were found to express GDNF, WNT1, and R-spondin 1 as well as pro-angiogenesis and immunomodulatory cytokines. In mice, IFNγ-stimulated MSC(M) injection after radiation decreased the loss of acinar cells, decreased the formation of fibrosis, and increased salivary production. ConclusionsMSC (M) from previously treated HNC patients can be expanded for auto-transplantation and are functionally active. Furthermore IFNγ-stimulated MSC(M) express proteins implicated in salivary gland regeneration. This study provides preliminary data supporting the feasibility of using autologous MSC(M) from HNC patients to treat RT-induced salivary dysfunction

Marrow-Derived Autologous Stromal Cells for the Restoration of Salivary Hypofunction (MARSH): A pilot, first-in-human study of interferon gamma–stimulated marrow mesenchymal stromal cells for treatment of radiation-induced xerostomia

Background aimsXerostomia, or the feeling of dry mouth, is a significant side effect of radiation therapy for patients with head and neck cancer (HNC). Preliminary data suggest that mesenchymal stromal/stem cells (MSCs) can improve salivary function. We performed a first-in-human pilot study of interferon gamma (IFNγ)-stimulated autologous bone marrow–derived MSCs, or MSC(M), for the treatment of radiation-induced xerostomia (RIX). Here we present the primary safety and secondary efficacy endpoints. MethodsA single-center pilot clinical trial was conducted investigating the safety and tolerability of autologous IFNγ-stimulated MSC(M). The study was conducted under an approved Food and Drug Administration Investigational New Drug application using an institutional review board–approved protocol (NCT04489732). Patients underwent iliac crest bone marrow aspirate and MSC(M) were isolated, cultured, stimulated with IFNγ and cryopreserved for later use. Banked cells were thawed and allowed to recover in culture before patients received a single injection of 10 × 106 MSC(M) into the right submandibular gland under ultrasound guidance. The primary objective was determination of safety and tolerability by evaluating dose-limiting toxicity (DLT). A DLT was defined as submandibular pain >5 on a standard 10-point pain scale or any serious adverse event (SAE) within 1 month after injection. Secondary objectives included analysis of efficacy as measured by salivary quantification and using three validated quality of life instruments. Quantitative results are reported as mean and standard deviation. ResultsSix patients with radiation-induced xerostomia who had completed radiation at least 2 years previously (average 7.8 years previously) were enrolled in the pilot study. The median age was 71 (61–74) years. Five (83%) patients were male. Five patients (83%) were treated with chemoradiation and one patient (17%) with radiation alone. Grade 1 pain was seen in 50% of patients after submandibular gland injection; all pain resolved within 4 days. No patients reported pain 1 month after injection, with no SAE or other DLTs reported 1 month after injection. The analysis of secondary endpoints demonstrated a trend of increased salivary production. Three patients (50%) had an increase in unstimulated saliva at 1 and 3 months after MSC(M) injection. Quality of life surveys also showed a trend toward improvement. ConclusionsInjection of autologous IFNγ-stimulated MSC(M) into a singular submandibular gland of patients with RIX is safe and well tolerated in this pilot study. A trend toward an improvement in secondary endpoints of salivary quantity and quality of life was observed. This first-in-human study provides support for further investigation into IFNγ-stimulated MSC(M) injected in both submandibular glands as an innovative approach to treat RIX and improve quality of life for patients with HNC.

Bone Marrow Biopsy, Does CT Guidance Add Value?

Background: Bone marrow aspirate and biopsies (BMAB) remain a corner stone in the diagnosis and management of various hematologic disorders. Specialized testing, such as cytogenetics, immunophenotypic and molecular analysis performed on these specimens, plays a crucial role in diagnosis and staging of Leukemias/Lymphomas, as well as in finding out molecular targets for potential therapeutic benefits. Thus, inadequate, and poor-quality specimens can limit making a precise diagnosis and providing optimal treatment. At our institution, we recognized that a substantial number of non-CT guided BMAB were being reported as suboptimal, although diagnosis was being made. Also, recently more CT guided (CTG) BMAB are being implemented. A drawback with CTG BMAB is scheduling delays, that can potentially result in treatment delays, and fatal outcomes especially in conditions like Acute Leukemias. Additionally, there is concern of higher cost associated with CT guided BMAB. Therefore, we performed a retrospective analysis on BMAB procedures performed at our institution between January 2019 and April 2022. Purpose:Evaluate and compare the quality of BMAB procedures performed with and without CT guidance (blind procedure) at our institution, and their respective waiting times.Identify potential areas for improvement.Provide data to help guide in choosing the most appropriate procedure. Methods: We retrospectively reviewed all adult BMAB procedures performed at our institution between January 2019 and April 2022, including patient demographics. Pathology reports were reviewed for adequacy of aspirate smears and core biopsies. Samples were deemed inadequate even if one of bone marrow aspirate or core or clot specimens were described by the pathologist as "suboptimal", "inadequate", "fragmented", "crushed", "hemodiluted", "no spicules", or "scant". Waiting period for the procedure was defined as the duration of time (in days) from the date of placing an order for biopsy (CTG or blind) to the date of procedure. CTG BMAB are typically performed by intervention radiologists (IR) while bind BMAB are usually performed by Oncologists (Fellows or attendings) at our institutions. Results: A total of 469 posterior iliac crest bone marrow aspirate and biopsy procedures were performed. More patients underwent BMAB procedure under CT guidance (61% vs 39% for non-CTG). More CTG BMAB samples were categorized as adequate (69%), compared to the non CTG (55%), (p=0.02). No statistical difference was noted in patient's age (p=0.639) or gender (p=.22) or Body mass index (BMI) (p=2.05) of CTG and non CTG groups (see table). The mean waiting period was longer for CTG procedure (27 days +/- 20 (0-135), compared to non CTG (15 days +/- 17 (0-105) (p=0.031). Although our pathologist described a sample as "suboptimal", a definitive diagnosis was still able to be made in all cases using the available bone marrow clot, aspirate and core specimens. Conclusion/Interventions: CTG BMAB procedures were more likely to result in adequate samples when compared to non-CTG procedures, however with longer waiting period and higher costs. So, we propose that obtaining non-CTG BMAB will improve cost effectiveness and reduce the wait times. To improve the adequacy of non CTG BMAB, we propose some interventions: 1. Provide an organized training during fellowship that includes theory followed by simulation, and practice-based training under supervision. 2. Discuss ICSH (International Council for Standardization in Hematology) guidelines for bone marrow adequacy criteria with the providers. For example, the length of a core biopsy from an adult should be at least 2cm. As our study suggests significant scheduling delays with CTG BMAB, such orders when requested, may be considered as urgent procedures to avoid potential treatment delays and poor outcomes. Alternately, when an aggressive hematologic disorder like acute leukemia is suspected, blind approach may be undertaken for prompt diagnosis. Interestingly, BMI was not significantly different in the two groups. However, if locating physical landmarks is impaired by body habitus, then CT guided BMAB should be strongly considered. We would like to highlight the importance of having adequate personnel including nurse navigators, technicians who play a vital role in obtaining timely insurance clearance, and procedure scheduling. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Outcomes of iliac crest bone marrow aspirate injection for the treatment of recalcitrant Achilles tendinopathy.

Achilles tendinopathy is a common cause of posterior ankle and heel pain in both active and sedentary patients. Though the majority of patients respond to first-line non-operative management including activity modification, immobilization, orthotics, and physical therapy with stretching and eccentric strengthening, there is no consensus for patients who fail these treatments. We evaluate the role of iliac crest bone marrow aspirate (BMA) injections as a treatment option for recalcitrant cases. A retrospective chart review was conducted of patients with refractory Achilles tendinopathy treated with iliac crest BMA concentrate injection. Symptoms were assessed using the numeric rating system (NRS) pain score at the pre-operative visit and at six, 12, 24, and 48weeks postoperatively. Post-operative complications were recorded. A total of 15 patients (15 feet) with recalcitrant Achilles tendinopathy (5 insertional, 8 non-insertional, 2 combined) treated with iliac crest BMA concentrate injections were included in the study. Average age was 53.2years (range, 25 to 64), average BMI was 27.1kg/m2 (range, 18.4 to 34.4), and average duration of symptoms prior to BMA injection was 2.3years (range, 1 to 7). Pre-operatively, average NRS was 6.26 (95% CI, 5.04 to 7.49), with significant improvement at sixweeks (mean, 4.26; 95% CI, 2.94 to 5.59; p = 0.04), tenweeks (mean, 4.13; 95% CI, 2.91 to 5.35; p = 0.012), 24weeks (mean, 3.40; 95% CI, 2.05 to 4.75; p = 0.03), and 48weeks (mean, 2.60; 95% CI, 1.14 to 4.06; p = 0.007) post-operatively. Overall, there was trending improvement over the 48-week follow-up period, with a mean improvement in NRS of - 3.22 (95% CI, - 1.06 to - 5.38; p = 0.007) at final follow-up. There was no discernable difference between insertional and non-insertional tendinopathy, and there were no incidences of post-operative complications. Iliac crest BMA appears to be a safe, effective, and potentially lasting treatment option for patients with intractable, insertional and non-insertional Achilles tendinopathy. Patients demonstrated and maintained statistically significant decrease in NRS pain score post-operatively with no complications at the donor or injection site.

Core decompression with autologous bone marrow aspirate injection in humeral head osteonecrosis in adults with sickle cell disease.

Sickle cell disease is often associated with osteonecrosis of the humeral head with a risk of progression to humeral head collapse. The aim of this study was to evaluate the clinical outcome and the effect on the necrosis evolution of humeral head core decompression with autologous bone marrow aspirate injection in these patients. Forty shoulders in 23 patients were treated with core decompression with autologous concentrated iliac crest bone marrow aspirate injection. Patients were followed for a minimum of two years (limits from 2 to 10 years). Functional outcomes metrics included the simple shoulder test (STT) and subjective shoulder value (SSV) as well as assessment of radiographic progression of disease. Shoulder function improved significantly at final follow-up. The STT improved by 2.9 points (p = 0.001) and the SVV improved by 15 % (p = 0.001). However, based on radiologic features, the intervention did not halt the radiographic progression of disease nor did it result in healing of the lesion. The benefits were mainly clinical with diminished pain and functional improvement. Improvement in shoulder function was observed following core decompression and bone marrow aspirate injection in patients with sickle cell disease and early humeral head necrosis stages. The procedure should not be considered for grade 3 or greater disease and for asymptomatic cases whatever the grade. Given the lack of radiographic improvement observed in this study, further study in the context of prospective controlled trials should be undertaken before this intervention can be widely recommended.

OPTIMIZING EX-VIVO EXPANDED AUTOLOGOUS TREG INFUSIONS AND DISCOVERING THEIR FATE USING AN IN VIVO PRIMATE MODEL

Introduction: Cell Therapy with regulatory Tcells (Tregs) is a promising tool for tolerance induction in solid organ transplantation. Despite encouraging results in humans and monkeys, questions about Treg phenotype, function, survival and migration after in vivo infusion remain. To better understand Treg fate, we have used a CFSE-labeled tracking method for autologous, polyclonal expanded Tregs in cynomolgus macaques. Methods: Polyclonal Tregs were grown from sorted (CD4+CD25hi) PBMCs and ex vivo expanded on allogeneic CD40-expressing Bcells and cryopreserved. 10-30x106 Tregs/kg were thawed and labeled with CFSE dye, then Tregs were administered intravenously. Phenotype and function of Tregs were specified via flow cytometry and suppression assays prior to in vivo infusion into recipient cynomolgus macaques. Labeled Tregs were then tracked in vivo with flow cytometry on peripheral blood and iliac crest bone marrow aspirates over the first month. At the time of sacrifice (Day 30), lymphoid organs (thymus, mesenteric and inguinal lymph nodes, spleen) and bone marrow were harvested and analyzed. Results: The first cynomolgus macaque received 12x106 CFSE-labeled Tregs/kg that retained their phenotype (CD4+CD25hi, FoxP3+ expression ≥95%) and suppressive capacities (1:16 suppression titer) throughout the expansion and restimulation process. CFSE-labeled Tregs were detected in circulation at Day 0, 2, 3, 6, 9 and 14 and were undetectable by day 15 post infusion. Dividing CFSE-labeled Tregs were seen at day 2,6 and 9 in the bone marrow. At day 30, no CFSE-labeled cells were found in any tissues. Treg infusions are upcoming in 4 more cynomolgus macaques. Conclusion: CFSE labeling of autologous, ex vivo expanded polyclonal Tregs is a valid model that allows in vivo tracking of migration and survival of Treg infusions in cynomolgus macaques.

A Specific Mesenchymal Stem and Progenitor Cell (MSPC) Subpopulation with a Multi-Potent Gene Signature Is Transcriptionally Altered in the Setting of Myelodysplastic Syndrome (MDS) in Primary Human Bone Marrow Aspirates

Introduction: Myelodysplastic syndromes (MDS) are genetically diverse and heterogeneous diseases characterized by dysplasia and cytopenias and a dismal prognosis of ˂ 50% overall survival at 5-years. In vitro and in vivo experimental data have shown that the bone marrow microenvironment (BMME) may play a role in disease progression and, importantly, in murine models, transplantation of MDS to a healthy BMME was shown to mitigate disease. Mesenchymal Stem and Progenitor Cells (MSPCs), as part of the BMME, give rise to multiple non-hematopoietic progenitor cells and provide essential support to hematopoietic stem cells. MSPCs have also been shown to be functionally altered in patients with myeloid neoplasias. Murine studies have demonstrated distinct roles for specific subsets of bone marrow mesenchymal cells in myeloid malignancies. We hypothesized that specific subsets of human bone marrow MSPCs will play differential roles in the pathogenesis of MDS. Using flow-cytometry, high-throughput sequencing and gene set enrichment analysis (GSEA), we characterized human MSPCs in the bone marrow aspirates from patients with MDS and normal healthy controls (NBM). Methods: Mononuclear cells were isolated by iliac crest bone marrow aspiration from 10-healthy donors and 14-patients at the University of Rochester Medical Center. Within the non-hematopoietic (CD45-, CD235-), non-endothelial (CD31-) bone marrow compartment, we enriched and isolated 3 distinct-subpopulations of MSPCs based on cell-surface expression of CD271/NGFR, CD106/VCAM-1 and CD146/MCAM. Populations were defined as follows: CD271+/CD146- (CD271+), CD271+/CD146+/CD106+ (DPCD106+), and CD271+/CD146+/CD106- (DPCD106-). RNA-seq analysis was performed on each subpopulation to define transcriptional signatures (TS) and gene set enrichment patterns. Statistically significant differentially expressed genes (DEGs) were defined by fold-change ≥ ±1 and p-value ˂0.05. Results: We first set out to define differences in the TS and interrogate the function of MSPC populations in NBM. Principle component analysis (PCA) demonstrated the highest variance between the DPCD106+ and the CD271+ populations. The number of DEGs were also highest between the DPCD106+ and CD271+ populations (n=3,619 genes). GSEA identified 745 and 336 gene sets with positive enrichment in the DPCD106+ and CD271+ group, respectively, and illustrated that the DPCD106+ population was significantly enriched in gene sets involved in early embryonic developmental and "stem-like" pathways whereas the CD271+ population was enriched in cell cycling, DNA and chromosomal organization. In the setting of MDS, the mean relative frequency of MDS CD271+ nearly tripled (0.4230/0.1445; p-value 0.045). Compared to NBM, MDS DPCD106+ cells had the highest variance by PCA and the highest number of DEGs (n=560). GSEA identified 19-gene sets with significant enrichment in the MDS DPCD106+ group and, intriguingly, 12 (63%) were identical to gene sets enriched in the CD271+ group. Furthermore, of the 560 DEGs in the MDS DPCD106+ MSPCs, 300 were upregulated and, of those, 160 (53%) were identical to upregulated genes in the CD271+ NBM group including the acquisition of a proliferative signature. Altogether, this data suggests a switch in the TS of theDPCD106+ population in the setting of MDS. Importantly, this TS clustered MDS DPCD106+ from NBM, regardless of MDS risk category. Conclusion: We successfully characterized 3 subtypes of MSPCs in NBM and MDS. In NBM, we demonstrate that cell surface expression of CD271, CD146 and CD106 defined the most stem-like TS within the non-hematopoietic, non-endothelial bone marrow compartment. In the setting of MDS, the increase in population frequency of the CD271+ cells and the concomitant transcriptomic aberrations observed in the MDS derived DPCD106+ population support the hypothesis that specific MSPC populations have differential roles in MDS pathogenesis. Further, we identify a TS that discriminates MDS derived MSPCs from NBM irrespective of MDS-risk category. This suggests that alterations within specific MSPC populations may represent a unifying pathway in disease pathogenesis despite heterogeneity and genetic drivers intrinsic to the MDS clone. Thus, targeting the BMME represents a potentially novel therapeutic strategy aimed at mitigating disease and restoring normal hematopoiesis in patients with MDS. Disclosures Liesveld: Onconova: Other: Data safety monitoring board; Abbvie: Membership on an entity's Board of Directors or advisory committees. Scadden:Editas Medicine: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Bone Therapeutics: Consultancy; Clear Creek Bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Novartis: Other: Sponsored research; Fate Therapeutics: Consultancy, Equity Ownership; Red Oak Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Fog Pharma: Consultancy; Agios Pharmaceuticals: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; LifeVaultBio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Efficacy of Autogenous Bone Marrow Aspirate as a Fusion-promoting Adjunct to Anterior Cervical Discectomy and Fusion: A Single Center Retrospective Cohort Study.

BackgroundAutogenous iliac crest bone marrow aspirate (BMA) has been shown to be a safe osteobiological adjunct to anterior cervical discectomy and fusion (ACDF), but little evidence exists to support its superiority to traditional methods. The object of this study was to retrospectively evaluate two cohorts of patients undergoing ACDF – with or without the use of BMA – in an effort to better characterize the clinical and radiographic outcomes associated with the use of BMA in ACDF.MethodsThe charts of all patients undergoing ACDF with a collagen-hydroxyapatite (CHA) sponge, local vertebral autograft and a polyetheretherketone (PEEK) interbody graft with or without BMA by a single staff neurosurgeon between 2011 and 2016 were retrospectively reviewed. Post-operative dynamic plain films and CT scans for each patient were reviewed and each instrumented level was independently evaluated for fusion over time.ResultsA total of 203 cervical levels were instrumented in 92 patients (with BMA, 52 patients, 122 levels; without BMA, 40 patients, 81 levels). The mean radiographic follow-up period was 21.4 ± 18.4 months, over which time 154 of 203 (75.6%) instrumented cervical levels were found to have fused (BMA group, 93/122 segments fused [76.2%]; non-BMA group, 61/81 segments fused [75.3%], p = 1). Kaplan-Meier survival analysis demonstrated a higher probability of fusion at any given time point for the BMA group when compared with the non-BMA group (p < 0.001, log-rank test).ConclusionsBMA is a readily accessible, low-cost adjunct to ACDF that enhances the fusion rates seen with a CHA/PEEK allograft combination.

The Clinical Impact of Macrofocal Disease in Multiple Myeloma Differs Between Presentation and Relapse

IntroductionFunctional imaging of Multiple Myeloma (MM) is redefining our knowledge of disease patterns. A pattern, termed macrofocal MM (macro MM), is defined by the presence of focal lesions and the absence of significant intervening bone marrow (BM) infiltration. At presentation, macro MM constitutes a distinct disease entity likely being associated with a favorable prognosis, although current evidence to support this is limited. Following first-line therapy, macrofocal patterns of disease emerge also in patients that initially presented with classical MM. In these patients the systemic BM involvement disappears in follow up examinations during treatment whereas focal lesions persist. In a third scenario, macrofocal patterns occur at overt relapse representing a patchy type of MM progression (Figure 1). The prognostic impact of a macrofocal pattern at these various disease stages is largely unknown. Therefore, we analyzed the clinical outcome and biological features of macro MM at different treatment stages.Patients and Methods279 patients met the criteria of macro MM. Of those, 56 were at initial presentation, 48 at restaging following first line therapy, and 175 at relapse. Generally, macrofocal lesions were present in both positron emission tomography and magnetic resonance imaging. All first-line patients were treated with multi-agent induction therapy, autologous stem cell transplantation and received maintenance within prospective trials. Outcome results were compared to a set of cases with classical MM matched for age, gene-expression based (GEP) risk group, and treatment protocol.ResultsMacro MM at presentation is rare, constituting 6% of patients in the time period examined. The vast majority showed GEP-based low risk (94%). Age, Ig-type, and sex were not significantly different between macro MM and classical MM. With a median follow up of 8.6 years, only 10 of the macro MM patients relapsed. Compared to a matched-pair MM group, progression-free survival (PFS) and overall survival (OS) were significantly better in the macro MM group (P= 0.01 and 0.04 for PFS and OS, respectively). Thus macro MM at presentation constitutes a low risk form of MM.Focusing on the 10 macro MM cases who relapsed, no specific risk profile could be identified except >26 focal lesions on MRI was associated with a shorter PFS (P=0.04) but not with OS. Of note, although focal lesions frequently responded slowly, the time to response was not associated with outcome.To elucidate whether there are biological differences between MM cells in focal lesions and at differentially involved BM sites, we analyzed a set of 16 patients with paired samples from macrofocal lesions and iliac crest BM aspirates. No difference in a GEP based proliferation index was seen between the two sites. After correction for multiple testing we did not observe gene expression differences between them. A candidate gene study including a set of 27, myeloma relevant, adhesion molecules also did not reveal expression differences.In contrast to the situation at presentation, macrofocal patterns at restaging during initial therapy showed a 70% cumulative 24 months relapse incidence. The outcome of these cases was significantly worse in comparison to matched controls (P=0.02 and 0.02 for PFS and OS, respectively). Of note, all patients with macro MM showed an objective response at the time of imaging with 9 of 46 cases meeting the IMWG criteria for CR.Performing a similar analysis of patients with macro MM at relapse showed that 25% of patients presented with that pattern; a surprisingly high proportion. Extramedullary involvement was common (41%). Of note, 36% of patients repeatedly showed macrofocal patterns at subsequent relapses. PFS and OS at 2 years from macrofocal relapse were 24% and 39%, respectively. A matched group OS comparison was not possible since number of relapses and treatments were too different among the patients.ConclusionsMacro MM at presentation seems to be an early stage of MM with an excellent prognosis. In contrast, a macrofocal pattern at restaging is associated with poor prognosis and early relapse. At this disease stage residual focal lesions may represent drug resistant clones. At overt relapse a macrofocal pattern was frequently seen, highlighting the need to integrate advanced imaging tools into the standard work up and indicating an important confounder of standard minimal residual disease diagnostics in MM. [Display omitted] DisclosuresBarlogie:Signal Genetics: Patents & Royalties. Davies:Janssen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Morgan:Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Bristol Meyers: Consultancy, Honoraria; Janssen: Research Funding; Univ of AR for Medical Sciences: Employment.

Ream content a stem cell source for bone defects

Bone grafts are normally used for the treatment of bone defects and non-union fractures, and the most common donor site is the iliac crest. The reamer-irrigator-aspirator (RIA) is an innovative technology to obtain osseous particles during intramedullary reaming in femur fractures. Discarded RIA can provide abundant native bone marrow mesenchymal stem cells (BM-MSCs) compared to the iliac crest. Autograft obtained from the use of RIA shows osteogenic potential at least equal to the iliac crest autograft with less donor site morbidity. The disadvantages of using the iliac crest including small volume, invasive harvesting, pain for the patient, makes RIA-harvested autograft bone preferable because it has good properties and is amenable for use as a sole alternative to the iliac crest graft or as a graft expander in conjunction with autologous iliac crest bone marrow aspirate.

Outcomes of Iliac Crest Bone Marrow Aspirate Injection for the Treatment of Recalcitrant Non- Insertional Achilles Tendinitis

Category: Sports Introduction/Purpose: Non-insertional Achilles tendinitis is a common cause of posterior ankle and heel pain in active and sedentary patients. Though the majority of patients respond to first-line non-operative management including activity modification, immobilization, orthotics, and physical therapy with tendon stretching, there is no consensus for patients that fail these treatments. We evaluate the role of iliac crest bone marrow aspirate concentrate (BMAC) as a safe and effective treatment option for recalcitrant cases. Methods: A retrospective chart review was conducted of patients with greater than 12 months of non-insertional Achilles tendinitis symptoms despite appropriate conservative treatment. Each patient had BMA harvested from the iliac crest, concentrated by centrifugation, and then injected into the Achilles tendon. Symptoms were assessed using the Visual Analogue Scale (VAS) pain score, collected at the preoperative office visit and at 6 weeks, 12 weeks, and 24 weeks after the procedure. Co- morbidities, concurrent procedures, and complications were also recorded. Results: A total of 21 patients (22 feet) were treated with iliac crest BMA concentrate injections. Preoperatively, the average VAS pain score was 6.8 (SD 2.0). Postoperatively, the average VAS was 4.0 (SD 2.5) at 6 weeks, 2.7 (SD 2.1) at 12 weeks, and 2.2 (SD 2.1) at 24 weeks. At 24 weeks postoperatively, there was a statistically significant decrease VAS score from baseline (p &lt; 0.001), with a mean decrease by 4.6 (SD 3.1). There were no adverse effects reported at the surgical site or donor site morbidity at the iliac crest such as wound infection, hematoma, or persistent pain. Conclusion: Iliac crest BMAC appears to be a safe, effective, and potentially lasting treatment option for patients with intractable, non-insertional Achilles tendinitis. Patients demonstrated a statistically significant decrease in VAS pain score postoperatively with no complications at the donor or injection site.

Revision Arthrodesis in the Foot and Ankle

Category: Ankle Arthritis Introduction/Purpose: Arthrodesis is a common surgical procedure used to manage arthritis and deformity in the foot and ankle. Nonunion or failure of the bone to fuse, is a known possible outcome in any arthrodesis surgery and often presents as persistent pain and swelling after an arthrodesis procedure. Published rates of nonunion in the foot and ankle literature range from 0 to 47 % depending on the patient population and joint involved. Multiple factors can contribute to developing a nonunion including; arthrodesis location, fixation method, tobacco use, diabetes, infection and others. Methods: The case logs of three fellowship trained foot and ankle surgeons were reviewed from January 2007 to September 2014. ICD-9 code 733.82 (nonunion fracture), 998.89 (miscellaneous complication), and a word search for nonunion in case logs were used to identify subjects. The clinical charts were reviewed for patient factors incuding: diabetes, inflammatory arthropathy, tobacco use, history of infection, prior nonunion elsewhere in the body, neuropathy, Charcot arthropathy, post-traumatic arthritis, and prior attempt at revision arthrodesis at the same clinical site. Surgical records were reviewed to identify location of the nonunion, type of instrumention, use of structural allograft, use of cancellous autograft (CA), use of iliac crest bone marrow aspirate (ICBMA) and use of orthobiologics such as bone morphogenetic protein (BMP) during the revision arthrodesis. Successful revision was defined as radiographic union in the final x-ray taken during follow-up by the attending orthopaedic surgeon and radiologist. Results: Eighty-two cases of revision arthrodesis were identified. The overall nonunion rate during revision arthrodesis was 23%. The average length of follow up was 16 months. Univariate analysis was performed on the patient and surgical factors to determine influence on nonunion rates. Of these factors, statistical analysis identified only neuropathy and prior attempts at revision as significant risks (P&lt; .05) for persistent nonunion. Odds ratio calculated based on previous attempts at revision arthrodesis found a 2.8 fold increase in the risk of failure for each previous attempt at revision. Charcot arthropathy approached significance at p=.051, but was limited by its small sample size (n=2). Smoking, location, use of orthobiologics and other clinical factors did not prove significant. Please see table for further details. Conclusion: Revision arthrodesis for nonunion in the foot and ankle can be highly successful (77%) under a variety of patient and surgical conditions. Neuropathy is a significant patient risk factor for persistent nonunions and is important to identify even in the non-diabetic patient. As the number of attempts at revisions increases there is a subsequent 3-fold increase in the risk of persistent nonunion.

Clinical and Operative Factors Related to Successful Revision Arthrodesis in the Foot and Ankle.

Arthrodesis is a common operative procedure used to manage arthritis and deformity in the foot and ankle. Nonunion is a possible and undesirable outcome in any arthrodesis surgery. Rates of nonunion in the foot and ankle literature range from 0% to 47% depending on the patient population and joint involved. Multiple factors can contribute to developing a nonunion including location, fixation method, tobacco use, diabetes, infection, and others. The case logs of 3 foot and ankle surgeons were reviewed from January 2007 to September 2014 to identify nonunion arthrodesis revision cases. The patient factors reviewed included diabetes, inflammatory arthropathy, tobacco use, history of infection, nonunion elsewhere, neuropathy, Charcot arthropathy, posttraumatic arthritis, and prior attempt at revision arthrodesis at the same site. Operative records were reviewed to identify location of the nonunion, instrumention, use of allograft or autograft bone, use of iliac crest bone marrow aspirate (ICBMA) and use of orthobiologics such as bone morphogenetic protein (BMP) during the revision arthrodesis. Successful revision was defined as radiographic union on the final radiograph during follow-up. Eighty-two cases of revision arthrodesis were identified with an average follow-up of 16 months. The overall nonunion rate was 23%. Neuropathy and prior attempts at revision were identified as significant risks (P <.05) for persistent nonunion. Odds ratio calculated based on previous attempts at revision arthrodesis found a 2.8-fold increase in the risk of failure for each attempt at revision. Revision arthrodesis for nonunion in the foot and ankle was successful (77%) under a variety of patient and operative conditions. Neuropathy was a significant patient risk factor for persistent nonunions, and we believe it is important to identify even in the nondiabetic patient. As the number of attempts at revisions increases, there is a subsequent 3-fold increase in the risk of persistent nonunion. Level IV, case series.

Natural hydroxyapatite as a bone graft extender for posterolateral spine arthrodesis.

The aim of this retrospective analysis of spinal fusion, was to document the clinical, functional, and radiological outcomes with a local bone graft plus the highly osteoconductive hydroxyapatite, bio-derived Orthoss®, with or without bone marrow aspirate. Forty seven patients submitted to spinal posterolateral fusion were operated for four major indications: scoliosis in young patients (11), degenerative spine (18), lumbosacral transitional anomalies in young adults (14), and spine trauma (four). Sixteen patients had more than four levels fused. In addition to spinal decompression and instrumented fusion, autologous bone grafts from the excised lamina were augmented with Orthoss® granules in a 1:1 ratio. In addition iliac crest bone marrow aspirate was used in 70% of the patients. The results were assessed clinically in terms of pain, and return to school or professional activities were checked at three, six, and 12months following surgery with a mean follow-up of 20months. In scoliotic patients, correction of the major angle was evaluated from one to fouryears after surgery. Pain persistence was reported only in four cases, after threemonths after surgery. A functional recovery was noted in almost all patients groups within these threemonths. Progressive bone formation with evidence of bone fusion masses were already observed at sixmonths. No fusion failure was observed. Local bone enhanced by an osteoconductive long-term stable scaffold, used with and without bone marrow aspirate, led to successful fusion in all patients by sixmonths while functional recovery was reported already within three to six months.

Comparison of fracture site callus with iliac crest bone marrow as the source of plastic-adherent cells

Background: Red marrow has been described as the main source of mesenchymal stem cells although its aspiration and isolation from bone marrow was reported to have significant donor site morbidity. Since secondary bone healing occurs through formation of callus as the result of proliferation and differentiation of mesenchymal stem cells, callus may become alternative source for mesenchymal stem cells. In this study, we compared the number of plastic-adherent cells from fracture site callus and bone marrow of iliac crest after two and four weeks of culture. Methods: Sixteen New Zealand rabbits were fracturized at the femoral shaft. Then, these rabbits were taken care. After two weeks of fracturization, 3 mL iliac crest bone marrow aspiration and callus extraction of eight rabbits were cultured (group I). The other eight rabbits were treated equally after four weeks of fracturization (group II). Simultaneously, the cultures were observed after one and two weeks. Four weeks later, they were harvested. Cells were counted using Neubauer hemocytometer. The average number of cells between the sources and groups were statistically analyzed using the unpaired t-test. Results: In group I, there were 2.6±0.1 x 104 cells in the culture of iliac crest bone marrow aspirate and 2.5 ± 0.1 x 104 cells in culture of callus extract from fracture site (p=0.34). In group II, there were 2.7 ± 0.1 x 104 cells and 2.1 ± 0.1 x 104 cells, respectively (p &lt; 0.001). Conclusion: Fracture site callus at the second week post-fracturization may be potential as source of plastic-adherent cells compared with iliac crest bone marrow.

Docetaxel, carboplatin, trastuzumab, and bevacizumab (TCH+B) for early-stage HER2/neu(+) breast cancer and bone marrow micrometastases.

TPS655 Background: Bone marrow (BM) micrometastases confer a poor prognosis to early-stage breast cancer (BC) and HER-2/neu(+) BC is a high-risk phenotype. We have shown that BM-associated stromal cells support the survival of metastatic BC in microenvironmental niches and this activity is dependent on VEGF and CXCL-12. Therefore, the growth of nascent tumor cell clusters in BM might be impeded by disrupting stromal cells or their activating ligands, such as VEGF. There is also a positive association between HER-2/neu and VEGF expression in BC, thereby implicating VEGF in the aggressive phenotype of HER-2/neu overexpression. These observations support the use of combination therapies directed against both HER-2/neu and VEGF for the treatment of HER-2/neu(+) BC. We therefore hypothesize that an anti-VEGF agent (bevacizumab) given with docetaxel, carboplatin and trastuzumab (TCH) will eradicate the BM of HER-2/neu(+) BC micrometastases by disrupting stromal cell support within the micrometastatic BM niche. Methods: Subjects with AJCC Stage I-III HER2/neu(+) BC will undergo a single iliac-crest BM aspirate and biopsy. If the BM is positive for CK+ micrometastases, up to 17 patients will receive TCH+B therapy (docetaxel at 75 mg/m2 IV and carboplatin at AUC 6 IV given every 3 weeks for 6 cycles; trastuzumab at 4 mg/kg IV loading dose, then 2mg/kg IV every week; bevacizumab at 15 mg/kg IV every 3 weeks). Four weeks after the 6th cycle of therapy, a repeat BM aspiration and biopsy will be performed to score for response of BC micrometastases to therapy. Patients with a complete clinical response in BM will continue with trastuzumab every 3 weeks at 6 mg/kg to a total of 1 year. Patients with persistent micrometastases will receive both trastuzumab and bevacizumab every 3 weeks to a total of 1 year of therapy. These patients will then have a repeat BM aspirate and biopsy at the end of the 1 year to score for final response. The primary objective of this clinical trial is to determine clinical response against BC micrometastases in BM. The second objective is to investigate the specific contribution of VEGF and CXCL-12 signaling to BM support of micrometastatic BC cells.

Combination cell therapy for the treatment of acute myocardial infarction

Over the past 10 years, several studies have been carried out to determine whether the infusion of peripheral blood (PBSC) or bone marrow-derived cell products (mononuclear, CD34, CD133 or ALDH cells) may improve cardiac function, after myocardial infarction (MI). The hypothesis behind these studies has been that infusion of a product containing endothelial progenitor cells (ECs)mayenhance the formation of newblood vessels. Results have shown thatwhile the infusion of these products is safe and feasible, the effect on left ventricular function has been negligible ormodest. The poor responses observed could be related to timing, type andnumber of cells used, route of administration or other variables [1,2]. The formation of blood vessel requires in addition to ECs, other cellular and molecular components, including pericytes, cytokines, chemokines and extracellular matrix molecules (ECM). Pericytes share functional properties and a gene-expression profile with mesenchymal stem cells (MSC). In turn, MSC produce and release angiogenic growth factors and ECM which contribute to the formation of a mature and stable blood vessel [3]. Based on these notions, we hypothesize that a combination, rather a single, cell product consisting of a source of ECs and pericytes could be more effective in promoting the formation of new blood vessels. This phase I clinical study was designed to investigate safety and feasibility after the intracoronary infusion to MI patients of a combination cell product consisting of bone marrow-derived mononuclear cell (BMMNC, a source of ECs) and MSC (a source of pericytes, angiogenic growth factors and ECM). Ten patients were enrolled to be treated with the combination cell product and five patients were used as controls. Table 1 shows main baseline characteristics of study and control patients. Study patients had a primary and secondary iliac crest bone marrow aspiration, 3–4 days after angioplasty and 17±7 days after primary aspiration, respectively. Bone marrow aspirations were not performed to control patients. For preparation of MSC and MNC, the methods previously published were used [4]. The combination cell product for infusion contained 7.5×10 MSC and 7.5×10 MNC (the content of EC-like cells in MNC measured as CD45 cells expressing antigens CD34, CD133 or CD144, was 3.5±1.1%.) After assessment of lot-release criteria (USP sterility, endotoxin, mycoplasma and Gram's stain), the combination cell product was intracoronary infused to study patients. After infusion, coronary angiography, ECG, chemistry panel, complete blood count (CBC) and Troponin I was performed. The average time between onset of MI and angioplasty was 15.7± 14 h. Bone marrow aspiration to initiate the preparation of BM-MSC was performed 3.7±1.8 days after PCI. In turn the cell product or placebo solution was infused 17.2±3.8 and 14.3±1.2 days after MI onset, respectively (Table 1). Intracoronary cell infusion probed to be safe and feasible and did not result in ischemia or infarction in the treated areas. Troponin I remained normal in all subjects at 24 h and 14 days post-infusion. Left Ventricular Ejection Fraction (LVEF)was evaluated in both study and control patients using quantitative gated SPECT at months 1 and 3. Echocardiography was utilized to evaluate ejection fraction and wall motion abnormalities. Table 2A shows data for LVEF at baseline and three months after cell/placebo infusion. No significant changes in LVEF or wall motion (not shown) were detected in either group. Assessment of myocardial ischemiawas performed using non-gated SPECT images myocardial perfusion summed stress score (SSS), summed rest score (SRS) and summed difference score (SDS). As seen in Table 2B, in the group of study patients no differences were observed between SSS and SRS at baseline and month 3 after cell infusion. However, this was not the case for SDS since in all treated patients, baseline scores were considerable higher than those at month 3. In turn, in control patients SSS, SRS and SDS values were comparable at baseline and month 3 after placebo infusion. This phase I studywas designed to assess the feasibility and safety of the intracoronary infusion of a combination cell product to MI patients. Feasibility was demonstrated by the procedural ability to initiate MSC manufacturing 3.7±1.8 days after angioplasty. In turn, the safe preparation and infusion of the cell products were confirmed by the inclusive fulfillment of release criteria and by the absence of adverse clinical events, up to 6 months after cell infusion. These observations corroborate results of our previous animal study showing safe and workable use of the combination cell product [5].

High abundance of CD271+ multipotential stromal cells (MSCs) in intramedullary cavities of long bones

Aspiration of iliac crest bone marrow (ICBM) remains the most frequent technique used in harvesting multipotential stromal cells (MSCs) for bone regeneration. Although this tissue type is easily accessed by a surgeon, it has a low frequency of MSCs, which is significant given the high cell numbers required for bone regeneration strategies. Lipoaspirates possess higher MSC frequencies, albeit cells with a differentiation profile less suited to orthopaedic interventions. Intra-medullary cavities of long bones have previously been shown to harbour MSCs in animals, however evaluation of their frequency, differentiation capacity and phenotype in humans had not previously been performed.Long bone fatty bone marrow (LBFBM) was collected prior to harvesting bone graft. Basic cellular compositions of donor-matched LBFBM and ICBM aspirates, including the numbers of CD34+ hematopoietic stem cells and CD31+ endothelial cells, were similar. MSCs were enumerated using colony-forming-unit-fibroblast assays and flow cytometry for the presence of a resident LBFBM CD45−/low CD271+ MSC population and revealed a trend for higher MSC numbers (average 5 fold, n=6) per millilitre of LBFBM compared to donor-matched ICBM. Functional characteristics of resident MSCs, including their growth rates, differentiation potentials and surface phenotypes (CD73+CD105+CD90+) before and after culture-amplification, were similar. Enhanced numbers of MSCs could be recovered following brief enzymatic treatment of solid fragments of LBFBM.Our findings therefore reveal that the intramedullary cavity of the human femur is a depot of MSCs, which, although closely associated with fat, have a differentiation profile equivalent to ICBM. This anatomical site is frequently accessed by the orthopaedic/trauma surgeon and aspiration of the intramedullary cavity represents a ‘low-tech’ method of harvesting potentially large numbers of MSCs for regenerative therapies and research.This article is part of a Special Issue entitled: Interactions Between Bone, Adipose Tissue and Metabolism.

The use of the reamer-irrigator-aspirator to harvest mesenchymal stem cells

The scarcity of mesenchymal stem cells (MSCs) in iliac crest bone marrow aspirate (ICBMA), and the expense and time in culturing cells, has led to the search for alternative harvest sites. The reamer-irrigation-aspirator (RIA) provides continuous irrigation and suction during reaming of long bones. The aspirated contents pass via a filter, trapping bony fragments, before moving into a 'waste' bag from which MSCs have been previously isolated. We examined the liquid and solid phases, performed a novel digestion of the solid phase, and made a comparative assessment in terms of number, phenotype and differentiation capacity with matched ICBMA. The solid fraction from the filtrate was digested for 60 minutes at 37° C with collagenase. Enumeration was performed via the colony-forming unit fibroblast (CFU-F) assay. Passage (P2) cells were differentiated towards osteogenic, adipogenic and chondrogenic lineages, and their phenotypes assessed using flow cytometry (CD33, CD34, CD45, CD73, CD90, and CD105). MSCs from the RIA phases were able to differentiate at least as well as those from ICBMA, and all fractions had phenotypes consistent with other established sources. The median number of colonies for the three groups was: ICBMA = 8.5 (2 to 86), RIA-liquid = 19.5 (4 to 90), RIA-solid = 109 (67 to 200) per 200 μl. The mean total yield of cells for the three groups was: ICBMA = 920 (0 to 4275), RIA-liquid = 114,983 (16,500 to 477,750), RIA-solid = 12,785 (7210 to 28 475). The RIA filtrate contains large numbers of MSCs that could potentially be extracted without enzymatic digestion and used for bone repair without prior cell expansion.

A Comparative Gene Expression Study of CD3+ T-Cells In Bone Marrow From Patients with Chronic ITP and Healthy Controls by DNA Microarray.

AbstractAbstract 1445 Background:The pathophysiology of ITP (immune thrombocytopenia) is more complex than initially believed. The importance of regulatory T-cells, platelet specific cytotoxic T-cells, suppression of megakaryocyte proliferation and maturation, functional thrombopoietin deficiency and inappropriately low bone marrow production of platelets has been unraveled over the last decade. T-cells are important in all forms of autoimmunity including ITP. However, very little is known about the T-cell activity in organs where platelets and their precursors are targeted by effectors of the immune system. To provide new insights into the pathophysiology of ITP, we performed a DNA microarray analysis on isolated bone marrow derived T-cells from chronic ITP patients and healthy controls. Patients and Method:Bone marrow was collected from 6 chronic ITP-patients (3 males and 3 females, mean age 39.5 years) and 6 healthy controls (3 males and3 females, mean age 33.8 years) through iliac crest bone marrow aspiration. T-cells were isolated by immunomagnetic cell sorting (Miltenyi Biotec, Surrey, UK). RNA was prepared using the Chomczynski method (Chomczynski and Sacchi, 1987), followed by RNeasy minielute clean-up (Qiagen, Hilden, Germany). RNA was amplified from 20 ng of starting total RNA with the Ovation RNA Amplification System V2 (NuGEN Technologies, Inc.), and cDNA was synthesized using the Encore Biotin Module kit (NuGEN Technologies, Inc.). After standard labeling, each sample was hybridized to an Affymetrix U133Plus 2.0 Human Genome array (Santa Clara, CA). Results:We identified 397 differently regulated genes in the bone marrow derived T-cells between ITP patients and controls by the DNA microarray analysis (P-value <0.05 and fold change ± 1.3). Some of the regulated genes have previously been implicated in ITP, such as integrin alpha 4 (ITGA4 or CD49D or VLA-4), Fas ligand (FASLG) and cytotoxic lymphocyte-associated 4 (CTLA-4). Furthermore, several apoptotic genes were lower expressed in ITP patients, such as Fas apoptotic inhibitory molecule 2 (FAIM2), BCL2-like 11 (BCL2L11), BCL2-like 13 (BCL2L13), peptidyl-tRNA hydrolase 2 (PTRH2) and the enzyme inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta (IKBKB).Integrin alpha 6, (ITGA6) and the adhesion molecule endothelial cell adhesion molecule (ESAM) were higher expressed in ITP patients compared to controls and are important for T-cell adhesion and migration or “homing” of lymphocytes to different lymphoid organs and inflammatory sites. Conclusion:We have identified a number of regulated genes in T-cells from bone marrow that differed between patients with ITP and healthy controls. The importance of several of the identified genes has already been implicated in ITP, proving the usefulness of this strategy. The identification of novel regulated genes may provide new insights into the physiology and pathophysiology of ITP and for autoimmunity in general. Disclosures:No relevant conflicts of interest to declare.