Synthetic peptides of IL-1Ra and HSP70 have anti-inflammatory activity on human primary monocytes and macrophages: Potential treatments for inflammatory diseases.

Chronic inflammatory diseases are increasing in developed societies, thus new anti-inflammatory approaches are needed in the clinic. Synthetic peptides complexes can be designed to mimic the activity of anti-inflammatory mediators, in order to alleviate inflammation. Here, we evaluated the anti-inflammatory efficacy of tethered peptides mimicking the interleukin-1 receptor antagonist (IL-1Ra) and the heat-shock protein 70 (HSP70). We tested their biocompatibility and anti-inflammatory activity in vitro in primary human monocytes and differentiated macrophages activated with two different stimuli: the TLR agonists (LPS + IFN-γ) or Pam3CSK4. Our results demonstrate that IL-1Ra and HSP70 synthetic peptides present a satisfactory biocompatible profile and significantly inhibit the secretion of several pro-inflammatory cytokines (IL-6, IL-8, IL-1ß and TNFα). We further confirmed their anti-inflammatory activity when peptides were coated on a biocompatible material commonly employed in surgical implants. Overall, our findings support the potential use of IL-1Ra and HSP70 synthetic peptides for the treatment of inflammatory conditions.

Chronic Glucocorticoids Consumption Triggers and Worsens Experimental Alzheimer's Disease-Like Pathology by Detrimental Immune Modulations.

INTRODUCTION: Among the risk factors identified in the sporadic forms of Alzheimer's disease (AD), environmental and lifestyle elements are of growing interest. Clinical observations suggest that stressful events can anticipate AD onset, while stress-related disorders can promote AD. Here, we tested the hypothesis that a chronic treatment with glucocorticoids is sufficient to trigger or exacerbate AD molecular hallmarks. METHODS: We first validated a rat model of experimental chronic glucocorticoids (GC) consumption (corticosterone [CORT] in drinking water for 4 weeks). Then, to evaluate the consequences of chronic GC consumption on the onset of amyloid-ß (Aß) toxicity, animals chronically treated with GC were intracerebroventricularly injected with an oligomeric solution of Aß25-35 (oAß) (acute model of AD). We evaluated AD-related cognitive deficits and pathogenic mechanisms, with a special emphasis on neuroinflammatory markers. RESULTS: Chronic CORT consumption caused the inhibition of the nonamyloidogenic pathways, the impairment of Aß clearance processes and the induction of amyloidogenic pathways in the hippocampus. The principal enzymes involved in glucocorticoid receptor activation and Tau phosphorylation were upregulated. Importantly, the AD-like phenotype triggered by chronic CORT was analogous to the one caused by oAß. These molecular commonalities across models were independent from inflammation, as chronic CORT was immunosuppressive while oAß was pro-inflammatory. When chronic CORT consumption anticipated the induction of the oAß pathology, we found a potentiation of neuroinflammatory processes associated with an exacerbation of synaptic and memory deficits but also an aggravation of AD-related hallmarks. DISCUSSION/CONCLUSION: This study unravels new functional outcomes identifying chronic CORT consumption as a main risk factor for AD and suggests that glucocorticoid-based therapies should be prescribed with caution in populations with AD risk.

Glucocorticoid receptors signaling impairment potentiates amyloid-ß oligomers-induced pathology in an acute model of Alzheimer's disease.

Dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis occurs early in Alzheimer's disease (AD), associated with elevated circulating glucocorticoids (GC) and glucocorticoid receptors (GR) signaling impairment. However, the precise role of GR in the pathophysiology of AD remains unclear. Using an acute model of AD induced by the intracerebroventricular injection of amyloid-ß oligomers (oAß), we analyzed cellular and behavioral hallmarks of AD, GR signaling pathways, processing of amyloid precursor protein, and enzymes involved in Tau phosphorylation. We focused on the prefrontal cortex (PFC), particularly rich in GR, early altered in AD and involved in HPA axis control and cognitive functions. We found that oAß impaired cognitive and emotional behaviors, increased plasma GC levels, synaptic deficits, apoptosis and neuroinflammatory processes. Moreover, oAß potentiated the amyloidogenic pathway and enzymes involved both in Tau hyperphosphorylation and GR activation. Treatment with a selective GR modulator (sGRm) normalized plasma GC levels and all behavioral and biochemical parameters analyzed. GR seems to occupy a central position in the pathophysiology of AD. Deregulation of the HPA axis and a feed-forward effect on PFC GR sensitivity could participate in the etiology of AD, in perturbing Aß and Tau homeostasis. These results also reinforce the therapeutic potential of sGRm in AD.

Stem cell therapy in early post-traumatic talus osteonecrosis.

PURPOSE: Avascular necrosis of the talus is one of the most notable complications associated with talar neck fractures with frequent evolution of the osteonecrosis into a difficult arthrodesis. We tested whether the injection of bone marrow mesenchymal stem cells (MSCs) could improve the repair process of the osteonecrosis. MATERIAL AND METHODS: Forty-five early (without collapse) post-traumatic talus osteonecroses (group 1; study group) were treated between 1995 and 2012 with percutaneous injection of progenitor cells (autologous bone marrow concentrate from the iliac crest). The number of MSCs transplanted in each ankle of group 1 was 124 × 103 cells (range 101 × 103 to 164 × 103 cells). The evolution of these osteonecroses treated with autologous bone marrow implantation was compared with the evolution of a control group of 34 talar osteonecroses without collapse and treated with only core decompression (group 2; control group) between 1985 and 1995. The outcome was determined by progression in radiographic stages to collapse, by the need of arthrodesis, and by the time to successfully achieve fusion for patients who needed arthrodesis. RESULTS: For the 45 ankles with autologous concentrate bone marrow grafting, collapse frequency was lower (27%, 12 among 45 versus 71%, 24 among 34; odds ratio 0.1515, 95% CI 0.0563-0.4079; P = 0.0002) and follow-up showed longer duration of survival before collapse or arthrodesis, compared to 34 ankles of the control patients with core decompression alone. Furthermore, the time to successfully achieve fusion after arthrodesis was significantly shorter in patients treated with bone marrow progenitors as compared with the other ankles, which had core decompression alone. CONCLUSION: In our study the early conservative surgical treatment with autologous bone marrow grafting improved the natural course of the disease as compared with core decompression alone.

Single-stage treatment of infected tibial non-unions and osteomyelitis with bone marrow granulocytes precursors protecting bone graft.

PURPOSE: Infected non-unions present a clinical challenge, especially with risk of recurrent infection. Bone marrow contains granulocyte precursors identified in vitro as colony forming units-granulocyte macrophage (CFU-GM) have a prophylactic action against infection. We therefore tested the hypothesis that bone marrow concentrated granulocytes precursors added to a standard bone graft could decrease the risk of recurrence of infection when single-stage treatment of infected tibial non-unions is performed with bone graft. METHODS: During a single-stage procedure 40 patients with infected tibial non-union received a spongious bone graft supercharged with granulocytes precursors after debridement (study group). A control group (40 patients) was treated in a single stage with local debridement and standard bone graft obtained from the iliac crest. The antibiotic therapy protocol was the same (60 days) in the two groups. CFU-GM progenitors were harvested from bone marrow aspirated on the opposite iliac crest of the site where the cancellous bone was obtained. Union (radiographs and CT scan), a recurrence of clinical infection, and need for subsequent surgery were evaluated. RESULTS: Thirty-eight (95%) patients who received graft supercharged with granulocytes precursors achieved successful union without recurrence of infection during the seven-year follow-up versus 28 (70%) control patients; for the control group the mean graft resorption volume was 40%, while no bone graft resorption was found for the study group. CONCLUSION: Supercharging the cancellous bone graft with bone marrow granulocytes precursors protect the site of infected non-union from recurrence of infection and bone resorption of the graft.

Subchondral stem cell therapy versus contralateral total knee arthroplasty for osteoarthritis following secondary osteonecrosis of the knee.

PURPOSE: Total knee arthroplasty (TKA) implanted in patients with secondary osteonecrosis (ON) related to corticosteroids have relatively poor outcome (20% revision rate) at a mean follow-up of only eight years. With the hypothesis that subchondral bone marrow injection might improve knees in these patients, we evaluated 30 patients who had bilateral knee osteoarthritis with severe joint space narrowing and received TKA in one knee and subchondral bone marrow concentrate injection in the contralateral knee. MATERIAL AND METHODS: A prospective randomized controlled clinical trial was carried out in 60 knees of 30 patients (mean age 28 years, 18-41) who presented bilateral osteoarthritis secondary to knee ON related to corticosteroids in relation with different severe medical conditions. During the same anesthesia, one knee received TKA; for the other knee, a bone marrow graft containing an average of 6500 MSCs/mL (counted as CFU-F, range 3420 to 9830) was delivered to the subchondral bone of the femur and tibia. The length of anesthesia related to each procedure (bone marrow aspiration and subchondral injection of concentrated bone marrow versus total knee arthroplasty) was measured. Peri-operative outcomes, morbidity, complications, and safety of the two procedures were compared. Subsequent admissions for revision surgery were identified. At the most recent follow-up (average of 12 years, range 8 to 16 years), clinical outcomes of the patient (Knee Society score) were obtained along with radiological imaging outcomes (MRIs for knees with subchondral bone marrow injection). RESULTS: Anesthesia related to the TKA side was longer than for the cell therapy group. Medical and surgical complications were more frequent after TKA. A higher number of thrombophlebitis was observed on the side with TKA (15%) versus none on the side with cell therapy (0%). At the most recent follow-up (average of 12 years, range 8 to 16 years), six (out of 30) TKA knees needed subsequent surgery versus only one with cell therapy. The Knee Score had improved and remained similar in the TKA and cell therapy groups (respectively 80.3 points ± 11 versus 78.3 ± 23); 21 patients preferred the knee with cell therapy and 9 preferred the knee with TKA. Knees with cell therapy had improvement on cartilage and bone marrow lesions observed at the site of bone marrow subchondral injection. CONCLUSIONS: Subchondral autologous bone marrow concentrate was an effective procedure for treating young patients with knee osteoarthritis following secondary ON of the knee related to corticosteroids with a lower complication rate and a quicker recovery as compared with TKA.

Cell therapy versus simultaneous contralateral decompression in symptomatic corticosteroid osteonecrosis: a thirty year follow-up prospective randomized study of one hundred and twenty five adult patients.

PURPOSE: Symptomatic osteonecrosis related to corticosteroids has a high risk of progression to collapse in absence of treatment. The purposes of this study were to evaluate the results of autologous bone marrow grafting of the symptomatic hip in adult patients with osteonecrosis and to compare the results with core decompression alone in the contralateral symptomatic hip. MATERIALS AND METHODS: A total of 125 consecutive patients (78 males and 47 females) with bilateral osteonecrosis (ON) and who had both hips symptomatic and at the same stage on each side (stage I or II) were included in this study from 1988 to 1998. The volume of osteonecrosis was measured with MRI in both hips; the smaller size ON was treated with core decompression, and the contralateral hip with the larger ON was treated with percutaneous mesenchymal cell (MSC) injection obtained from bone marrow concentration. The average total number of MSCs (counted as number of colony forming units-fibroblast) injected in each hip was 90,000 ± 25,000 cells (range 45,000 to 180,000 cells). RESULTS: At the most recent FU (average 25 years after the first surgery, range 20 to 30 years), among the 250 hips included in the study, 35 hips (28%) had collapsed at the most recent follow-up after bone marrow grafting, and 90 (72%) after core decompression (CD). Ninety-five hips (76%) in the CD group underwent total hip replacement and 30 hips (24%) in the bone marrow graft group (p < 0.0001). Hips undergoing only CD were approximately three times more likely to undergo a primary THA (odds ratio: 10.0278; 95% CI: 5.6117 to 17.9190; p < 0.0001) as compared with hips undergoing an initial bone marrow grafting. For the 90 hips treated with bone marrow injection and without collapse, the mean volume of repair evaluated by MRI at the most recent follow-up was 16.4 cm3 (range 12 to 21 cm3) corresponding to a decrease of the pre-operative average volume from 22.4 cm3 (range 35-15 cm3) to 6 cm3 (range 12-0 cm3); as percentage of the volume of the femoral head, the decrease moved from 44.8 to 12%. CONCLUSION: Core decompression with bone marrow injection improved the outcome of the disease as compared with core decompression alone in the same patient.

History of concentrated or expanded mesenchymal stem cells for hip osteonecrosis: is there a target number for osteonecrosis repair?

PURPOSE: Despite multiple possible treatments, the risk of collapse remains the main problem of osteonecrosis. Heart failure (HF). In an effort to address the reverse this issue, curative strategies with regenerative medicine are increasingly being considered. The aim of this technology is to halt or reverse progression of the disease to collapse. MATERIAL AND METHODS: The pioneering report by Hernigou published in 2002 was the first pilot study suggesting that injection of bone marrow stem cells was a safe approach able to improve osteonecrosis in patients with early stages. Since then, an impressive number of studies and trials employing unselected BM-derived cells (1000 the last 2 years) showed that delivery of those cells to the site of osteonecrosis during core decompression was somehow able to ameliorate the patient with osteonecrosis. In order to translate the promise of this cell therapy into better clinical benefit, many questions need to be addressed. In this review, we therefore analyzed current clinical experience of the literature and our experience of 4000 cases to address these questions and particularly the number of cells that should be injected. RESULTS: After almost 20 years of clinical research in this field, we are still far from having drawn conclusions on the number of cells we should inject in regenerating hip osteonecrosis. Findings are difficult to interpret due to heterogeneity of causes of osteonecrosis, as well as differences in the cells count, sample quality, and stages of osteonecrosis. The authors address specific issues, as cell quality, cell numbers, volume of osteonecrosis, concentration of cells, and ex vivo expansion. Bone marrow mesenchymal stem cells are supposed to be "functionally competent," but are collected from the bon, marrow of patients with diseases and risk factors of osteonecrosis. The recipient organ (bone osteonecrosis) is a tissue where several alterations have already occurred. These questions are addressed in this review. CONCLUSION: In this review, we analyzed current clinical experience regarding cell therapy and address issues that should be a guide for future cell-based therapeutic application in osteonecrosis.

Allografts supercharged with bone-marrow-derived mesenchymal stem cells possess equivalent osteogenic capacity to that of autograft: a study with long-term follow-ups of human biopsies.

PURPOSE: Bone-marrow-derived mesenchymal stem cells (BM-MSCs) have been proposed to enhance bone formation in allografts. However, it is not known whether a combination of MSCs, contained in bone marrow concentrate (BMC) and structural allograft could be better than an allograft without MSCs and equivalent to a femoral head autograft in terms of histologic bone formation and long-term cellularity in the graft. After ten years of follow-up, three types of grafts: those initially loaded with BM-MSCs; dead, irradiated allografts; autografts. MATERIALS AND METHODS: Twenty patients received acetabular grafting during hip surgery and subsequently underwent femoral hip revision eight to 13 years later (average 10 years). Revision surgery was for reasons other than graft failure. These 20 patients had received eight allografts initially loaded with BM-MSCs: six dead irradiated allografts and six autografts. The number of MSCs present in the three types of graft were evaluated at the time of initial surgery and at revision. New bone formation associated in the acetabular graft was assessed by histology and calculated as a percentage of total available bony area. RESULTS: At the most recent follow-ups (average 10 years), concentration of MSCs in allografts previously loaded with BM-MSCs was higher than that found in autografts. There were low or no MSCs found in uncharged allografts. New-bone-formation analysis showed that allografts loaded with BM-MSCs produced more new bone (35 %; range 20-50 %) compared with either uncharged allografts (9 %; range 2-15 %) or autografts (24 %; range 12-32 %). CONCLUSIONS: Our observations with allografts charged with BM-MSCs provides evidence in support of a long-term benefit of supercharging bone allografts with autologous BM-MSCs.

Deficiency in either COX-1 or COX-2 genes does not affect amyloid beta protein burden in amyloid precursor protein transgenic mice.

Epidemiologic studies indicate that chronic use of non-steroidal anti-inflammatory drugs (NSAIDs) is associated with a lower risk for developing Alzheimer's disease (AD). Because the primary mode of action of NSAIDs is to inhibit cyclooxygenase (COX) activity, it has been proposed that perturbed activity of COX-1 or COX-2 contributes to AD pathogenesis. To test the role of COX-1 or COX-2 in amyloid deposition and amyloid-associated inflammatory changes, we examined amyloid precursor protein (APP) transgenic mice in the context of either COX-1 or COX-2 deficiency. Our studies showed that loss of either COX-1 or COX-2 gene did not alter amyloid burden in brains of the APP transgenic mice. However, one marker of microglial activation (CD45) was decreased in brains of COX-1 deficient/APP animals and showed a strong trend in reduction in COX-2 deficient/APP animals. These results suggest that COX activity and amyloid deposition in brain are likely independent processes. Further, if NSAIDs do causally reduce the risks of AD, then our findings indicate that the mechanisms are likely not due primarily to their inhibition on COX or γ-secretase modulation activity, the latter reported recently after acute dosing of ibuprofen in humans and nonhuman primates.

Osteogenic progenitors in bone marrow aspirates have clinical potential for tibial non-unions healing in diabetic patients.

PURPOSE: There is a significantly higher incidence of delayed unions, non-unions, and increased healing time in diabetic patients compared with non-diabetic patients. Studies suggest that diabetics suffer from deficiencies of pancreatic stem/progenitor cells, and a clinically relevant question arises concerning the availability and functionality of progenitor cells obtained from bone marrow of diabetics for applications in bone repair. METHODS: We have evaluated the cellularity and frequency of osteogenic mesenchymal stem cells (MSCs) in bone marrow from 54 diabetic patients (12 with type 1 and 42 with type 2) with tibial non-unions. These patients were treated with bone marrow MSCs (BM-MSCs) delivered in an autologous bone marrow concentrate (BMC). Clinical outcomes and marrow cellularity were compared to 54 non-diabetic, matched patients with tibial non-unions also treated with BMC. RESULTS: After adjusting for age and sex, no differences were identified with respect to bone marrow cellularity and MSC number among the diabetic and non-diabetic groups and both groups received approximately the same number of MSCs on average. BMC treatment promoted non-union healing in 41 diabetic patients (76 %) and 49 non-diabetic patients (91 %), but the non-diabetic patients healed more quickly and produced a larger volume of callus. CONCLUSION: We recommend that diabetic patients be treated with an increased number of progenitor cells by increasing the bone marrow aspiration volume. We also anticipate a need to extend the time of casting and non-weight bearing for diabetic patients as compared with non-diabetic patients.

Local transplantation of bone marrow concentrated granulocytes precursors can cure without antibiotics infected nonunion of polytraumatic patients in absence of bone defect.

PURPOSE: Infected, long bone non-unions present a significant clinical challenge. New and alternative therapies are needed to address this problem. The purposes of this study were to compare the number of circulating granulocyte-macrophage colony-forming units (CFU-GM) in the peripheral blood of polytraumatic patients with infected tibial non-unions and in the peripheral blood of control patients with the hypothesis that their number was decreased in polytraumatic patients; and to treat their infection without antibiotics and with local transplantation of bone marrow concentrated granulocytes precursors. METHODS: Thirty (18 atrophic and 12 hyperthrophic ) infected tibial non-unions (without bone defect) that occurred after open fractures in polytraumatic patients were treated without antibiotics and with percutaneous injection of autologous bone marrow concentrate (BMC) containing granulocytes precursors (CFU-GM). CFU-GM progenitors were assessed in the bone marrow aspirate, peripheral blood, and fracture site of these patients. The number of these progenitors was compared with the CFU-GM progenitors of control patient samples (healthy donors matched for age and gender). Outcome measures were: timing of union, callus formation (radiographs and CT scan), and recurrence of clinical infection. RESULTS: As compared to control patients, the number of CFU GM derived colonies was lower at peripheral blood in patients with infected nonunions. The bone marrow graft injected in nonunions contained after concentration 42 621 ± 20 350 CFU-GM-derived colonies/cc. Healing and cure of infection was observed at six months for 25 patients and at one year follow up for 30 patients. At the median ten year follow-up (range: 5 to 15), only one patient had clinical recurrent infection after healing (between 6 months and last follow-up). CONCLUSION: The peripheral blood of these polytraumatic patients with infected nonunions had a remarkable decrease in CFU-GM-derived colonies as compared with normal controls. Local transplantation of concentrated CFU-GM-derived colonies aspirated from bone marrow allowed cure of infection and healing without antibiotics.

Percutaneous injection of bone marrow mesenchymal stem cells for ankle non-unions decreases complications in patients with diabetes.

PURPOSE: Clinical studies in diabetic patients have demonstrated that there is a high incidence of complications in distal tibia and ankle fracture treatments. One strategy to mitigate issues with wound healing and infection in diabetic patients is to use a percutaneous technique in which autologous, bone marrow-derived, concentrated cells are injected at the site of non-unions. METHODS: Eighty-six ankle non-union in diabetic patients were treated with bone marrow mesenchymal stem cells (BM-MSCs) delivered in an autologous bone marrow concentrate (BMC). Clinical outcomes of the 86 diabetic non-union patients treated with BMC were compared with 86 diabetic matched non-unions treated with a standard bone iliac crest autograft. RESULTS: Treatment with BMC promoted non-union healing in 70 among 86 diabetic patients (82.1 %) with a low number of complications. Of the 86 diabetic patients treated with iliac bone graft, 53 (62.3 %) had healing; major complications were observed: 5 amputations, 11 osteonecroses of the fracture wound edge and 17 infections. CONCLUSIONS: In diabetic patients with ankle non-unions, treatment with BM-MSCs from bone marrow concentrate may be preferable in view of the high risks of major complications after open surgery and iliac bone grafting, and improved healing rates compared with standard iliac bone autograft treatment.

The degree of BCR and NFAT activation predicts clinical outcomes in chronic lymphocytic leukemia.

B-cell antigen receptor (BCR)-mediated signaling plays a critical role in chronic lymphocytic leukemia (CLL) pathogenesis and gives an in vitro survival advantage to B cells isolated from patients with unfavorable prognostic factors. In this study, we undertook to elucidate the signaling intermediates responsible for this biologic alteration. In responding cells only, in vitro BCR engagement triggers global phosphorylation of Syk, activation of phospholipase Cγ2, and intracellular calcium mobilization, reflecting competency of BCR signaling. The calcium-calcineurin-dependent transcription factor NFAT2 is up-regulated and to some extent constitutively activated in all CLL B cells. In contrast, its DNA-binding capacity is enhanced on IgM stimulation in responding cells only. NFAT inhibition using the VIVIT peptide prevents induction of CD23 target gene and IgM-induced survival, converting responding cells to unresponsive status. At the opposite, ionomycin-induced NFAT activity allows survival of nonresponding cells. These results demonstrate that the functional heterogeneity relies on variability of protein levels establishing BCR-dependent thresholds and NFAT-dependent activation. Finally, status of the BCR-NFAT pathway for each patient reveals its relevance for CLL clinical outcome and points out to BCR-NFAT intermediates as promising functional therapeutic targets.

Regenerative therapy with mesenchymal stem cells at the site of malignant primary bone tumour resection: what are the risks of early or late local recurrence?

PURPOSE: There is concern that regenerative cell-based therapies at the site of malignant primary bone tumours could result in increased risk of local tumour recurrence. We therefore investigated the long-term risks for site-specific recurrences in patients who had received an autologous bone marrow derived mesenchymal stem cell suspension to improve healing at the host-to-allograft bone junction of the reconstruction after bone tumour resection. METHODS: A total of 92 patients were treated from 1993 to 2003 with bone marrow-derived mesenchymal stem cells after bone tumour resection. Patients were monitored for cancer incidence from the date of first operation (1993) until death, or until 31 December 2013. The mean follow-up time was 15.4 years (range ten to 20 years). The average number of MSCs returned to the patient was 234,000 MSCs ± 215,000. The primary outcome was to evaluate the risk of tumorigenesis recurrence at the cell therapy treatment sites with radiographs and/or MRIs. The relative risk of cancer recurrence was expressed as the ratio of observed and expected number of cases according to three different control populations. RESULTS: Thirteen recurrences were found at the treatment sites among the 92 patients. The expected number of recurrences based on incidence in the three cohort populations was between 15 and 20 for the same cancer, age and sex distribution. The standardized incidence ratio (equal to observed cancers divided by expected cancers) for the entire follow-up period and for all recurrences was between 0.65 and 0.86 (95 % CI 0.60-1.20). CONCLUSION: This study found no increased cancer local recurrence risk in patients after application of autologous cell-based therapy using bone marrow-derived mesenchymal stem cells at the treatment site after an average follow-up period of 15.4 years, ranging from ten to 20 years.

Biologic augmentation of rotator cuff repair with mesenchymal stem cells during arthroscopy improves healing and prevents further tears: a case-controlled study.

PURPOSE: The purpose of this study was to evaluate the efficiency of biologic augmentation of rotator cuff repair with iliac crest bone marrow-derived mesenchymal stem cells (MSCs). The prevalence of healing and prevention of re-tears were correlated with the number of MSCs received at the tendon-to-bone interface. METHODS: Forty-five patients in the study group received concentrated bone marrow-derived MSCs as an adjunct to single-row rotator cuff repair at the time of arthroscopy. The average number of MSCs returned to the patient was 51,000 ± 25,000. Outcomes of patients receiving MSCs during their repair were compared to those of a matched control group of 45 patients who did not receive MSCs. All patients underwent imaging studies of the shoulder with iterative ultrasound performed every month from the first postoperative month to the 24th month. The rotator cuff healing or re-tear was confirmed with MRI postoperatively at three and six months, one and two years and at the most recent follow up MRI (minimum ten-year follow-up). RESULTS: Bone marrow-derived MSC injection as an adjunctive therapy during rotator cuff repair enhanced the healing rate and improved the quality of the repaired surface as determined by ultrasound and MRI. Forty-five (100 %) of the 45 repairs with MSC augmentation had healed by six months, versus 30 (67 %) of the 45 repairs without MSC treatment by six months. Bone marrow concentrate (BMC) injection also prevented further ruptures during the next ten years. At the most recent follow-up of ten years, intact rotator cuffs were found in 39 (87 %) of the 45 patients in the MSC-treated group, but just 20 (44 %) of the 45 patients in the control group. The number of transplanted MSCs was determined to be the most relevant to the outcome in the study group, since patients with a loss of tendon integrity at any time up to the ten-year follow-up milestone received fewer MSCs as compared with those who had maintained a successful repair during the same interval. CONCLUSION: This study showed that significant improvement in healing outcomes could be achieved by the use of BMC containing MSC as an adjunct therapy in standard of care rotator cuff repair. Furthermore, our study showed a substantial improvement in the level of tendon integrity present at the ten-year milestone between the MSC-treated group and the control patients. These results support the use of bone marrow-derived MSC augmentation in rotator cuff repair, especially due to the enhanced rate of healing and the reduced number of re-tears observed over time in the MSC-treated patients.

Supercharging irradiated allografts with mesenchymal stem cells improves acetabular bone grafting in revision arthroplasty.

PURPOSE: The procedure of bone allografting associated with a reinforcement device is widely used for acetabulum revision. However in absence of biologic fixation of the allograft, failure of the reconstruction may occur. We made the hypothesis that it would be possible to load these grafts with bone marrow derived mesenchymal stem cells (MSC) to rescue the osteogenic capacity of an allogenic dead bone and therefore enhance incorporation of allografts with the host bone and decrease the number of failures related to the allograft. METHOD: We identified 60 patients who had undergone acetabular component revision for aseptic failure of cemented implants associated with massive periacetabular osteolysis and Paprosky type 3A or 3B classification (without pelvic discontinuity) between 1996 and 2001. The study group of 30 patients received MSCs in the allograft and at the host graft junction. The average total number of MSCs received by each patient was 195,000 cells (range 86,000-254,000 cells). The control group of 30 patients had no MSCs in the allograft. Patients were matched for the size of periacetabular osteolysis (Paprosky type 3A or 3B). We compared the evolution of the allografts and evaluated cup migration and revision of the hips as end points at a minimum of 12 years or until failure. RESULT: Better radiographic graft union rates and less allograft resorption were observed with allografts loaded with stem cells. Allograft resorption was significantly decreased in the group with allograft loaded with MSCs (1.2 cm(2) -range 0-2.3 cm(2)-of resorption on radiographs in the group with MSCs; versus 6 cm(2), range 2.1-8.5 cm(2) in the group without MSCs). The rate of mechanical failure was highest (p = 0.01) among the 30 patients with allograft without stem cells (9/30; 30 %) compared with no failures for patients with allograft loaded with stem cells. Revision of the cup was necessary in nine patients in the control group. No revision was performed in the 30 patients of the study group with MSCs. CONCLUSION: For acetabular defect reconstruction, loading the allograft with MSCs has resulted in a lower rate of failure as compared with allograft without MSCs.

Morbidity of graft harvesting versus bone marrow aspiration in cell regenerative therapy.

PURPOSE: Using bone marrow mesenchymal stem cells (MSCs) with aspiration from the iliac crest is commonly used in reconstructive orthopaedic surgery. Because bone marrow aspiration is a percutaneous technique, the morbidity as compared with the classical bone graft should be decreased. METHOD: Therefore in a retrospective review of 523 consecutive cases of bone marrow aspiration performed at the Henri Mondor Hospital from 1990 to 2006 for the treatment of fractures, minor and major complications were identified and compared to the number of complications observed during the same period with 435 classical iliac crest bone graft procedures performed for the same indications of treatment of fractures. Minor complications included superficial infections, superficial seromas, and minor haematomas. Major complications included herniation of abdominal contents through massive bone graft donor sites, vascular injuries, deep infections at the donor site, neurologic injuries, deep haematoma formation requiring surgical intervention or transfusion, and iliac wing fractures. RESULT: Bone marrow aspiration decreased significantly the number of complications as compared with harvesting classical iliac crest bone graft that was associated with significant morbidity. Adverse events were significantly lower (p < 0.01) in the 523 procedures with bone marrow aspiration as compared with the 435 bone iliac crest piece harvesting. This was true for anaemia (16 cases versus 87 cases), for early pain (six versus 152), persistent pain (two versus 21), neuralgia (three versus 11), minor complications (ten versus 56), and major complications (three cases versus 22 cases). CONCLUSION: In our series the number of complications with bone marrow aspiration was ten times less than the complications observed with the classical technique of bone piece harvesting from the iliac crest, and the complications were clearly less severe.

New preclinical porcine model of femoral head osteonecrosis to test mesenchymal stromal cell efficiency in regenerative medicine.

PURPOSE: In order to evaluate new therapeutic approaches to human osteonecrosis of the femoral head (ONFH), this study proposed to improve the existing animal model by developing a new surgically induced pig model. METHODS: First, ONFH was induced with an easy and minimally invasive technique: cryogenic insult with repeated freeze-thaw cycle. Then, to compare and improve the efficacy of this first method, we combined the cryogenic insult to vascular coagulation of the posterior circumflex vessels. RESULTS: Cryoinjury with repeated freeze-thaw cycle alone is sufficient to induce, three weeks postsurgery, a subchondral necrosis as confirmed by magnetic resonance imaging (MRI) and histological analysis. However, a bone regeneration began at four weeks and was complete at eight weeks. To optimise this result, we combined cryoinjury with posterior circumflex vessel coagulation and observed the persistence of ONFH, with progression to collapse at 14 weeks postinduction. CONCLUSIONS: Cryoinjury associated with partial vascular coagulation is sufficient to obtain localised and sustainable necrosis in the subchondral area of the femoral head, reproducing all stages of the human disorder. The co-analysis by MRI and histology allowed us to confirm that the classic T1- and T2-weighted hyposignal regeneration front around a fatty high T1-weighted signal observed by MRI indicate signs of induced osteonecrosis. Our results indicate that our pig model induces all stages of human ONFH, which can be followed by MRI, making it relevant for clinical trials.

Benefits of small volume and small syringe for bone marrow aspirations of mesenchymal stem cells.

PURPOSE: Aspirating bone marrow from the iliac crest using small volumes of 1-4 ml with a 10-ml syringe has been historically proposed for harvesting adult mesenchymal stem cells and described as a standard technique to avoid blood dilution. The disadvantage of repeated small aspirations is that there is a significantly increased time to harvest the bone marrow. However, it is not known if a large volume syringe can improve the rate of bone marrow aspiration without increasing blood dilution, thus reducing the quality of the aspirate. We compared the concentrations of mesenchymal stem cells obtained under normal conditions with two different size syringes. METHODS: Thirty adults (16 men and 14 women with a mean age of 49 ± 14 years) underwent surgery with aspiration of bone marrow from their iliac crest. Bilateral aspirates were obtained from the iliac crest of the same patients with a 10-ml syringe and a 50-ml syringe. Cell analysis determined the frequencies of mesenchymal stem cells (as determined by the number of colonies) from each size of syringe. The cell count, progenitor cell concentration (colonies/ml marrow) and progenitor cell frequency (per million nucleated cells) were calculated. All bone marrow aspirates were harvested by the same surgeon. RESULTS: Aspirates of bone marrow demonstrated greater concentrations of mesenchymal stem cells with a 10-ml syringe compared with matched controls using a 50-ml syringe. Progenitor cell concentrations were on average 300 % higher using a 10-ml syringe than matched controls using a 50-ml syringe (p < 0.01). CONCLUSIONS: In normal human donors, bone marrow aspiration from 30 patients demonstrated a reduced mesenchymal stem cell number in aspirates obtained using a larger volume syringe (50 ml) as compared with a smaller volume syringe (10 ml).