Characterization of three washing/decellularization procedures for the production of bioactive human micronized neural tissue (hMINT).

BACKGROUND: We developed a novel, injectable and decellularized human peripheral nerve-based scaffold, named Micronized Human Neural Tissue (hMINT), designed to be used as a supportive matrix for stem cell transplantation in the context of spinal cord injury (SCI). MATERIALS AND METHODS: Human donated sciatic nerves were micronized at liquid nitrogen temperature prior to decellularization using 3 different procedures of various harshness. hMINT were characterized in terms of particle size, DNA, sulfated glycosaminoglycans (sGAG) and growth factors content. To test the biocompatibility and bioactivity of the various preparations, we used a type of mesenchymal stromal cells (MSCs), termed MIAMI cells, which were placed in contact with hMINT to monitor cell attachment by confocal microscopy and gene expression by RT-qPCR in vitro. RESULTS: The content of DNA, sGAG and growth factors left in the product after processing was highly dependent on the decellularization procedure used. We demonstrated that hMINT are biocompatible and promoted the attachment and long-term survival of MIAMI cells in vitro. Finally, combination with hMINT increased MIAMI cells mRNA expression of pro-survival and anti-inflammatory factors. Importantly, the strongest bioactivity on MIAMI cells was observed with the hMINT decellularized using the mildest decellularization procedure, therefore emphasizing the importance of achieving an adequate decellularization without losing the hMINT's bioactivity. PERSPECTIVES AND CLINICAL SIGNIFICANCE: The capacity of hMINT/stem cells to facilitate protection of injured neural tissue, promote axon re-growth and improve functional recovery will be tested in an animal model of SCI and other neurodegenerative disorders in the future.

Growth Hormone-Releasing Hormone Receptor Antagonist Modulates Lung Inflammation and Fibrosis due to Bleomycin.

PURPOSE: Growth hormone-releasing hormone (GHRH) is a 44-amino acid peptide that regulates growth hormone (GH) secretion. We hypothesized that a GHRH receptor (GHRH-R) antagonist, MIA-602, would inhibit bleomycin-induced lung inflammation and/or fibrosis in C57Bl/6J mice. METHODS: We tested whether MIA-602 (5 µg or vehicle given subcutaneously [SC] on days 1-21) would decrease lung inflammation (at day 14) and/or fibrosis (at day 28) in mice treated with intraperitoneal (IP) bleomycin (0.8 units on days 1, 3, 7, 10, 14, and 21). Bleomycin resulted in inflammation and fibrosis around airways and vessels evident histologically at days 14 and 28. RESULTS: Inflammation (histopathologic scores assessed blindly) was visibly less evident in mice treated with MIA-602 for 14 days. After 28 days, lung hydroxyproline (HP) content increased significantly in mice treated with vehicle; in contrast, lung HP did not increase significantly compared to naïve controls in mice treated with GHRH-R antagonist. GHRH-R antagonist increased basal and maximal oxygen consumption of cultured lung fibroblasts. Multiple genes related to chemotaxis, IL-1, chemokines, regulation of inflammation, and extracellular signal-regulated kinases (ERK) were upregulated in lungs of mice treated with bleomycin and MIA-602. MIA-602 also prominently suppressed multiple genes related to the cellular immune response including those for T-cell differentiation, receptor signaling, activation, and cytokine production. CONCLUSIONS: MIA-602 reduced lung inflammation and fibrosis due to bleomycin. Multiple genes related to immune response and T-cell functions were downregulated, supporting the view that MIA-602 can modulate the cellular immune response to bleomycin lung injury.

Supplemental Fixation of Inner Graft Limbs in All-Inside, Quadrupled, Single-Tendon Anterior Cruciate Ligament Reconstruction Graft Construct Yields Improved Biomechanical Properties.

PURPOSE: To compare the time-zero load to failure of a quadrupled, single-tendon, all-inside anterior cruciate ligament (ACL) reconstruction graft construct with (supplemented) and without the incorporation of inner-limb whipstitch sutures (control) into a tibial suspensory fixation button. METHODS: Eight matched pairs of peroneus longus tendons were prepared according to a quadrupled, all-inside ACL soft-tissue graft technique with 1 side serving as a control and the contralateral side supplemented. The constructs were biomechanically tested for strain in the inner and outer limbs during a preconditioning protocol, single-cycle load to failure, and elongation of the whole construct. RESULTS: Ultimate load to failure was significantly higher in the supplemented group: 797.5 ± 49.6 N (95% confidence interval [CI], 763.13-831.87 N) versus 719.6 ± 69.6 N (95% CI, 671.38-767.82 N; P = .044). Less graft elongation at failure was observed in the supplemented group (3.1 ± 1.5 mm; 95% CI, 2.07-4.17 mm) versus the control group (21.0 ± 21.2 mm; 95% CI, 6.31-35.69 mm; P = .052). The number of grafts undergoing a 5-mm or greater change in length at failure was 1 of 8 in the supplemented group versus 5 of 8 in the control group (P = .038). CONCLUSIONS: Inner-limb supplemental tibial fixation results in higher time-zero load to failure and decreased graft elongation in a quadrupled, single-tendon, all-inside ACL reconstruction graft construct. CLINICAL RELEVANCE: The weak point of a single-tendon, quadrupled, all-inside ACL graft construct is the tendon-to-tendon suturing to secure the inner limbs of the graft. Adding supplemental fixation by incorporating the sutures from the inner limb to the tibial suspensory fixation button leads to a higher time-zero load to failure and decreased graft elongation.

Histologic, biomechanical, and biological evaluation of fan-folded iliotibial band allografts for anterior cruciate ligament reconstruction.

PURPOSE: The purpose of this study was to thoroughly characterize the fan-folded iliotibial band (FITB) allograft and compare it with anterior tibialis tendons (ATs) and native anterior cruciate ligaments (ACLs) to determine whether it measures up to those tissues. METHODS: We compared the histologic structure, tensile strength to failure, creep, and stress-relaxation properties of FITBs with those of ATs and ACLs. In vitro cytotoxicity and biocompatibility of FITBs were also compared with ATs. RESULTS: No structural difference was observed between the tissues studied. FITB ultimate tensile strength (3,459 ± 939 N) was not significantly different (P > .9999) from ultimate tensile strength of ATs (3,357 ± 111 N) and was significantly greater (P = .0005) than that of ACLs (886 ± 254 N). No significant difference (P > .9999) was observed in the increase in length resulting from creep testing between FITBs (9.5 ± 3.0 mm) and ATs (9.7 ± 4.0 mm). During stress-relaxation testing, FITBs reached 181 ± 46 N, which was not significantly different (P > .9999) from ATs (166 ± 40 N). Finally, we showed that cytotoxicity of FITBs and ATs was negligible. In vitro biocompatibility of FITBs and ATs was very good, whereas FITBs had a higher propensity to favor the attachment and infiltration of cells that proliferated for at least 4 weeks on their contact. CONCLUSIONS: We found that FITBs, ACLs, and ATs shared a similar structure made of aligned collagen fibers. No significant difference was observed between FITB and AT ultimate tensile strength, creep, and stress-relaxation viscoelastic properties. Ultimate tensile strength to failure of ACLs was lower than that of FITBs and ATs, whereas ACLs were superior to both FITBs and ATs during creep and stress-relaxation testing. FITBs and ATs showed low cytotoxicity and excellent biocompatibility in vitro, with a somewhat higher propensity of FITBs to favor cell attachment and infiltration over time. CLINICAL RELEVANCE: This study suggests that FITBs have the potential to perform as well as ATs for ACL reconstruction.

Basivertebral nerve ablation for the treatment of chronic low back pain in a community practice setting: 6 Months follow-up.

Background: Strong innervation of the vertebral endplates by the basivertebral nerve makes it an ideal target for ablation in the treatment of vertebrogenic low back pain with Modic changes. This data represents the clinical outcomes for 16 consecutively treated patients in a community practice setting. Methods: Basivertebral nerve ablations were performed on 16 consecutive patients by a single surgeon (WS) utilizing the INTRACEPT® device (Relievant Medsystems, Inc.). Evaluations were performed at baseline, 1 month, 3 months, and 6 months. The Oswestry Disability Index (ODI), Visual Analog Scale (VAS), and SF-36 were recorded in Medrio electronic data capture software. All patients (n = 16) completed the baseline, 1 month, 3 months, and 6 months follow-up. Results: The ODI, VAS, and SF-36 Pain Component Summary showed statistically significant improvements above minimal clinically important differences at 1 month, 3 months, and 6 months (all p values <0.05). Change in ODI pain impact declined 13.1 points [95% CI: 0.01,27.2] at one month from baseline, 16.5 points [95% CI: 2.5,30.6] at three months from baseline, and 21.1 points [95% CI: 7.0,35.2] six-months from baseline. SF-36 Mental Component Summary also showed some improvements, but with significance only at 3 months (p = 0.0091). Conclusions: Basivertebral nerve ablation appears to be a durable, minimally invasive treatment for the relief of chronic low back pain that can be successfully implemented in a community practice setting. To our knowledge, this is the first independently funded US study on basivertebral nerve ablation.

Viable cryopreserved human bone graft exhibit superior osteogenic properties in mandibular lateral augmentation.

Lack of bone volume to place dental implants is frequently a problem in the reconstruction of edentulous patients. Even though autografts are the gold standard for jaw regeneration, morbidity associated with the harvesting site stimulates the demand for other substitutes. The aim of this study is to characterize the incorporation and the osteogenic ability of a viable cryopreserved human bone graft (VC-HBG) in the mandibular augmentation in rats. Bone chips from fresh human vertebrae cadaveric donors were processed, cryoprotected and deep-frozen at - 80 °C maintaining its cell viability. A jaw augmentation model was used in 20 athymic nude rats allocated into 2 groups to either receive the VC-HBG or an acellular graft as control (A-HBG). The assessment of the grafts' incorporation was performed at 4 and 8 weeks by micro-CT, histomorphometry and immunohistochemistry. Bone volume gain was significantly higher for the VC-HBG group at both time points. At 4 weeks, the A-HBG group presented significantly higher mineral density, but at 8 weeks, the VC-HBG group showed significantly higher values than the A-HBG. There was no statistical difference between VC-HBG and A-HBG groups at 4-weeks for remaining graft particles, while at 8 weeks, the VC-HBG group showed significantly less graft remnants. Collagen I, osteopontin and tartrate-resistant acid phosphatase expression were significantly higher in the VC-HBG group at both time points, while osteocalcin expression was significantly higher in the VC-HBG group at 8-weeks compared to the A-HBG group. This experimental research demonstrated that the VC-HBG shows positive osteogenic properties, greater bone formation, higher rate of bone remodeling and a better overall incorporation in rats' mandibles compared to the A-HBG.

Basivertebral Nerve Ablation for the Treatment of Chronic Low Back Pain: A Scoping Review of the Literature.

BACKGROUND: Chronic low back pain is a leading cause of disability worldwide and its pathophysiology remains poorly understood, a problem exacerbated by the heterogeneity of the patient population with chronic low back pain. Although the intervertebral discs are often implicated in chronic low back pain, studies have demonstrated strong innervation of the vertebral endplates by the basivertebral nerve, therefore making it a possible target for ablation in the treatment of vertebrogenic chronic low back pain. OBJECTIVES: This work reviews the current evidence for the efficacy and safety of basivertebral nerve ablation as a treatment modality for chronic low back pain, and discusses the possible study biases and gaps in the current knowledge to provide insight on future research. STUDY DESIGN: The authors registered with the Center for Open Sciences and followed the Preferred Reporting Items for Systematic Reviews and Meta-analyses Extension for Scoping Reviews  (PRISMA-ScR). SETTING: A private clinic. METHODS: This study was performed in accordance with the following 5-stage methodological framework for scoping reviews: (i) identifying the research question; (ii) identifying relevant studies; (iii) selecting studies; (iv) charting the data; and (v) collating, summarizing and reporting the results. Three databases (PubMed, Web of Science, Embase) were searched using the keywords "basivertebral", "nerve", and "ablation". RESULTS: From March 2002 to March 2022, a total of 47 articles were identified, of which 12 were included in this scoping review, based on the exclusion criteria described in Table 1. LIMITATIONS: The limitations found were: • A very specific chronic pain population is typically utilized for this intervention. The inclusion criteria leave many who experience chronic low back pain ineligible for the procedure. • Study demographics need to be more diversified to truly represent the chronic low back pain population.• There is a lack of true control groups due to high crossover rates in published studies.• Very few high-level or long-term studies have been published.• Funding for many of the studies published on the subject is industry-led (Table 6). With an already limited amount of published research, a need for out-of-industry funding is required to avoid any possibility of bias. CONCLUSIONS: Current research has shown that basivertebral nerve ablation might be a promising treatment for chronic low back pain in patients exhibiting Modic type 1 or 2 endplate changes, while additional research on the association between Modic changes and low back pain is still needed to gain widespread use and acceptance of this new treatment modality. The introduction of new devices and a larger number of independent studies would greatly enhance the confidence in the outcomes reported with this treatment modality in order to ultimately benefit patients, clinicians, and society.

Improved Sensation Resulting From Spinal Cord Stimulation for the Treatment of Painful Diabetic Neuropathy: The Possible Role of Stochastic Resonance.

BACKGROUND: Painful diabetic neuropathy (PDN) is a progressive chronic pain condition that significantly affects the quality of life of patients with long-standing diabetes mellitus. Sensory deficits may result in falls, foot ulceration, and lower limb amputations. Recently, spinal cord stimulation (SCS) was studied for treatment of painful diabetic neuropathy. In addition to pain relief, we were surprised to discover that sensory improvements were also demonstrated. No mechanistic explanation has yet been offered to explain these findings. OBJECTIVES: Sensory improvements were observed in patients during the Senza-PDN clinical trial. Our objective was to offer a hypothesis to explain these results. STUDY DESIGN: The randomized, prospective, multicenter, open-label Senza-PDN clinical trial was aimed at documenting the value of 10 kHz SCS in addition to conventional medical management alone. We formulated an hypothesis to explain the neurologic improvement observed while using SCS in these study patients. SETTING: This work was conducted in a private clinical practice. RESULTS: SCS resulted in an overall decrease in pain for the enrolled PDN patients. An unexpected improvement in neurologic outcomes was also noticed at up to 12 months, which had never been reported before. We hypothesized that stochastic resonance mechanism could explain these sensory improvements. We believe that waveforms delivered to the spinal cord may have had the unexpected effect of creating noise-enhanced signal processing. LIMITATIONS: Further research will have to be performed to confirm the plausibility of the stochastic resonance hypothesis formulated. CONCLUSIONS: SCS might have unexpected benefits in patients with PDN beyond pain reduction. The Senza-PDN trial is the first to describe improved sensation in association with SCS. While the mechanism of action are still unknown, we hypothesize that noise-enhanced signal processing via stochastic resonance may explain these results. Stochastic resonance, or the benefit of additional randomness, should be further studied in the context of spinal cord stimulation. Further, SCS programming that optimizes for stochastic resonance should also be investigated for restoration of sensory and possibly even motor function.

EGF and bFGF pre-treatment enhances neural specification and the response to neuronal commitment of MIAMI cells.

AIMS: Multipotent mesenchymal stromal cells raise great interest for regenerative medicine studies. Some MSC subpopulations have the potential to undergo neural differentiation, including marrow isolated adult multilineage inducible (MIAMI) cells, which differentiate into neuron-like cells in a multi-step neurotrophin 3-dependent manner. Epidermal and basic fibroblast growth factors are often used in neuronal differentiation protocols for MSCs, but with a limited understanding of their role. In this study, we thoroughly assessed for the first time the capacity of these factors to enhance the neuronal differentiation of MSCs. MATERIALS AND METHODS: We have characterized MIAMI cell neuronal differentiation program in terms of stem cell molecule expression, cell cycle modifications, acquisition of a neuronal morphology and expression of neural and neuronal molecules in the absence and presence of an EGF-bFGF pre-treatment. RESULTS: EGF-bFGF pre-treatment down-regulated the expression of stemness markers Oct4A, Notch1 and Hes5, whereas neural/neuronal molecules Nestin, Pax6, Ngn2 and the neurotrophin receptor tyrosine kinase 1 and 3 were up-regulated. During differentiation, a sustained Erk phosphorylation in response to NT3 was observed, cells began to exit from the cell cycle and exhibit increased neurite-like extensions. In addition, neuronal ß3-tubulin and neurofilament expression was increased; an effect mediated via the Erk pathway. A slight pre-oligodendrocyte engagement was noted, and no default neurotransmitter phenotype was observed. Overall, mesodermal markers were unaffected or decreased, while neurogenic/adipogenic PPARγ2 was increased. CONCLUSION: EGF and bFGF pre-treatment enhances neural specification and the response to neuronal commitment of MIAMI cells, further increasing their potential use in adult cell therapy of the nervous system.

A Combinatorial Cell and Drug Delivery Strategy for Huntington's Disease Using Pharmacologically Active Microcarriers and RNAi Neuronally-Committed Mesenchymal Stromal Cells.

For Huntington's disease (HD) cell-based therapy, the transplanted cells are required to be committed to a neuronal cell lineage, survive and maintain this phenotype to ensure their safe transplantation in the brain. We first investigated the role of RE-1 silencing transcription factor (REST) inhibition using siRNA in the GABAergic differentiation of marrow-isolated adult multilineage inducible (MIAMI) cells, a subpopulation of MSCs. We further combined these cells to laminin-coated poly(lactic-co-glycolic acid) PLGA pharmacologically active microcarriers (PAMs) delivering BDNF in a controlled fashion to stimulate the survival and maintain the differentiation of the cells. The PAMs/cells complexes were then transplanted in an ex vivo model of HD. Using Sonic Hedgehog (SHH) and siREST, we obtained GABAergic progenitors/neuronal-like cells, which were able to secrete HGF, SDF1 VEGFa and BDNF, of importance for HD. GABA-like progenitors adhered to PAMs increased their mRNA expression of NGF/VEGFa as well as their secretion of PIGF-1, which can enhance reparative angiogenesis. In our ex vivo model of HD, they were successfully transplanted while attached to PAMs and were able to survive and maintain this GABAergic neuronal phenotype. Together, our results may pave the way for future research that could improve the success of cell-based therapy for HDs.

Human Bone Marrow-Derived Mesenchymal Stromal Cell-Seeded Bone Biomaterial Directs Fast and Superior Mandibular Bone Augmentation in Rats.

Atrophic maxillary ridges present a challenge in the field of oral implantology. Autologous bone is still considered the gold standard grafting material, but the increased morbidity and surgical complications represent a major drawback for its use. The aim of this study was to assess the efficacy of an off-the-shelf cell-seeded bone biomaterial for mandibular bone augmentation, compared to its acellular counterpart. We used a rat model to test the osteogenic properties of bone marrow-derived mesenchymal stromal cells (MSCs)-seeded bone microparticles compared to acellular bone microparticles alone. Rats were euthanized at 4 and 8 weeks, and results analyzed using micro-CT imaging, histology (H&E, Masson's Trichrome), histomorphometry and immunohistology (Tartrate-Resistant Acid Phosphatase-TRAP, Osteocalcin and human specific anti-mitochondria antibodies). Micro-CT analysis demonstrated that the cell-seeded biomaterial achieved significantly more bone volume formation at 4 weeks (22.75 ± 2.25 mm3 vs 12.34 ± 2.91 mm3, p = 0.016) and at 8 weeks (64.95 ± 5.41 mm3 vs 42.73 ± 10.58 mm3, p = 0.029), compared to the acellular bone microparticles. Histology confirmed that the cell-seeded biomaterial was almost completely substituted at 8 weeks, in opposition to the acellular biomaterial group. Immunohistochemical analysis showed a significantly higher number of TRAP and Osteocalcin positive cells at 4 weeks in the cell-seeded group compared to the acellular group, thereby demonstrating a higher rate of bone remodeling in the presence of MSCs. The grafted human cells remained viable and were detected up to at least 8 weeks, as observed using the human specific anti-mitochondria antibody. This off-the-shelf material available in unlimited quantities could therefore represent a significant advance in the field of mandibular bone augmentation by providing a larger volume of new bone formation in a shorter time.

Expression and characterization of trehalose biosynthetic modules in the adjacent locus of the salbostatin gene cluster.

The pseudodisaccharide salbostatin, which consists of valienamine linked to 2-amino-1,5-anhydro-2-deoxyglucitol, is a strong trehalase inhibitor. From our Streptomyces albus ATCC 21838 genomic library, we identified thirty-nine ORFs in a 40-kb gene cluster. Twenty-one genes are supposed to be a complete set of modules responsible for the salbostatin biosynthesis. Through sequence analysis of the gene cluster, some of the upstream gene products (SalB, SalC, SalD, SalE, and SalF) revealed functional resemblance with trehalose biosynthetic enzymes. On the basis of this rationale, we isolated the five genes (salB, salC, salD, salE, and salF) from the S. albus ATCC 21838 and cloned them into the expression vector pWHM3. We demonstrated the noticeable expression and accumulation of trehalose, using only the five upstream biosynthetic gene cluster of salbostatin, in the transformed Streptomyces lividans TK24. Finally, 490 mg/l trehalose was produced by fermentation of the transformant with sucrosedepleted R2YE media.

Marrow-isolated adult multilineage inducible cells embedded within a biologically-inspired construct promote recovery in a mouse model of peripheral vascular disease.

Peripheral vascular disease is one of the major vascular complications in individuals suffering from diabetes and in the elderly that is associated with significant burden in terms of morbidity and mortality. Stem cell therapy is being tested as an attractive alternative to traditional surgery to prevent and treat this disorder. The goal of this study was to enhance the protective and reparative potential of marrow-isolated adult multilineage inducible (MIAMI) cells by incorporating them within a bio-inspired construct (BIC) made of two layers of gelatin B electrospun nanofibers. We hypothesized that the BIC would enhance MIAMI cell survival and engraftment, ultimately leading to a better functional recovery of the injured limb in our mouse model of critical limb ischemia compared to MIAMI cells used alone. Our study demonstrated that MIAMI cell-seeded BIC resulted in a wide range of positive outcomes with an almost full recovery of blood flow in the injured limb, thereby limiting the extent of ischemia and necrosis. Functional recovery was also the greatest when MIAMI cells were combined with BICs, compared to MIAMI cells alone or BICs in the absence of cells. Histology was performed 28 days after grafting the animals to explore the mechanisms at the source of these positive outcomes. We observed that our critical limb ischemia model induces an extensive loss of muscular fibers that are replaced by intermuscular adipose tissue (IMAT), together with a highly disorganized vascular structure. The use of MIAMI cells-seeded BIC prevented IMAT infiltration with some clear evidence of muscular fibers regeneration.

Pharmacologically active microcarriers delivering BDNF within a hydrogel: Novel strategy for human bone marrow-derived stem cells neural/neuronal differentiation guidance and therapeutic secretome enhancement.

Stem cells combined with biodegradable injectable scaffolds releasing growth factors hold great promises in regenerative medicine, particularly in the treatment of neurological disorders. We here integrated human marrow-isolated adult multilineage-inducible (MIAMI) stem cells and pharmacologically active microcarriers (PAMs) into an injectable non-toxic silanized-hydroxypropyl methylcellulose (Si-HPMC) hydrogel. The goal is to obtain an injectable non-toxic cell and growth factor delivery device. It should direct the survival and/or neuronal differentiation of the grafted cells, to safely transplant them in the central nervous system, and enhance their tissue repair properties. A model protein was used to optimize the nanoprecipitation conditions of the neuroprotective brain-derived neurotrophic factor (BDNF). BDNF nanoprecipitate was encapsulated in fibronectin-coated (FN) PAMs and the in vitro release profile evaluated. It showed a prolonged, bi-phasic, release of bioactive BDNF, without burst effect. We demonstrated that PAMs and the Si-HPMC hydrogel increased the expression of neural/neuronal differentiation markers of MIAMI cells after 1week. Moreover, the 3D environment (PAMs or hydrogel) increased MIAMI cells secretion of growth factors (b-NGF, SCF, HGF, LIF, PlGF-1, SDF-1α, VEGF-A & D) and chemokines (MIP-1α & ß, RANTES, IL-8). These results show that PAMs delivering BDNF combined with Si-HPMC hydrogel represent a useful novel local delivery tool in the context of neurological disorders. It not only provides neuroprotective BDNF but also bone marrow-derived stem cells that benefit from that environment by displaying neural commitment and an improved neuroprotective/reparative secretome. It provides preliminary evidence of a promising pro-angiogenic, neuroprotective and axonal growth-promoting device for the nervous system. STATEMENT OF SIGNIFICANCE: Combinatorial tissue engineering strategies for the central nervous system are scarce. We developed and characterized a novel injectable non-toxic stem cell and protein delivery system providing regenerative cues for central nervous system disorders. BDNF, a neurotrophic factor with a wide-range effect, was nanoprecipitated to maintain its structure and released in a sustained manner from novel polymeric microcarriers. The combinatorial 3D support, provided by fibronectin-microcarriers and the hydrogel, to the mesenchymal stem cells guided the cells towards a neuronal differentiation and enhanced their tissue repair properties by promoting growth factors and cytokine secretion. The long-term release of physiological doses of bioactive BDNF, combined to the enhanced secretion of tissue repair factors from the stem cells, constitute a promising therapeutic approach.

Effects of gamma irradiation on the biomechanical properties of peroneus tendons.

PURPOSE: This study was designed to investigate the biomechanical properties of nonirradiated (NI) and irradiated (IR) peroneus tendons to determine if they would be suitable allografts, in regards to biomechanical properties, for anterior cruciate ligament reconstruction after a dose of 1.5-2.5 Mrad. METHODS: Seven pairs of peroneus longus (PL) and ten pairs of peroneus brevis (PB) tendons were procured from human cadavers. The diameter of each allograft was measured. The left side of each allograft was IR at 1.5-2.5 Mrad, whereas the right side was kept aseptic and NI. The allografts were thawed, kept wet with saline, and attached in a single-strand fashion to custom freeze grips using liquid nitrogen. A preload of 10 N was then applied and, after it had reached steady state, the allografts were pulled at 4 cm/sec. The parameters recorded were the displacement and force. RESULTS: The elongation at the peak load was 10.3±2.3 mm for the PB NI side and 13.5±3.3 mm for the PB IR side. The elongation at the peak load was 17.4±5.3 mm for the PL NI side and 16.3±2.0 mm for the PL IR side. For PL, the ultimate load was 2,091.6±148.7 N for NI and 2,122.8±380.0 N for IR. The ultimate load for the PB tendons was 1,485.7±209.3 N for NI and 1,318.4±296.9 N for the IR group. The ultimate stress calculations for PL were 90.3±11.3 MPa for NI and 94.8±21.0 MPa for IR. For the PB, the ultimate stress was 82.4±19.0 MPa for NI and 72.5±16.6 MPa for the IR group. The structural stiffness was 216.1±59.0 N/mm for the NI PL and 195.7±51.4 N/mm for the IR side. None of these measures were significantly different between the NI and IR groups. The structural stiffness was 232.1±45.7 N/mm for the NI PB and 161.9±74.0 N/mm for the IR side, and this was the only statistically significant difference found in this study (P=0.034). CONCLUSION: Our statistical comparisons found no significant differences in terms of elongation, ultimate load, or ultimate stress between IR and NI PB and PL tendons. Only the PB structural stiffness was affected by irradiation. Thus, sterilizing allografts at 1.5-2.5 Mrad of gamma irradiation does not cause major alterations in the tendons' biomechanical properties while still providing a suitable amount of sterilization for anterior cruciate ligament reconstruction.

Low Oxygen Modulates Multiple Signaling Pathways, Increasing Self-Renewal, While Decreasing Differentiation, Senescence, and Apoptosis in Stromal MIAMI Cells.

Human bone marrow multipotent mesenchymal stromal cell (hMSC) number decreases with aging. Subpopulations of hMSCs can differentiate into cells found in bone, vasculature, cartilage, gut, and other tissues and participate in their repair. Maintaining throughout adult life such cell subpopulations should help prevent or delay the onset of age-related degenerative conditions. Low oxygen tension, the physiological environment in progenitor cell-rich regions of the bone marrow microarchitecture, stimulates the self-renewal of marrow-isolated adult multilineage inducible (MIAMI) cells and expression of Sox2, Nanog, Oct4a nuclear accumulation, Notch intracellular domain, notch target genes, neuronal transcriptional repressor element 1 (RE1)-silencing transcription factor (REST), and hypoxia-inducible factor-1 alpha (HIF-1α), and additionally, by decreasing the expression of (i) the proapoptotic proteins, apoptosis-inducing factor (AIF) and Bak, and (ii) senescence-associated p53 expression and ß-galactosidase activity. Furthermore, low oxygen increases canonical Wnt pathway signaling coreceptor Lrp5 expression, and PI3K/Akt pathway activation. Lrp5 inhibition decreases self-renewal marker Sox2 mRNA, Oct4a nuclear accumulation, and cell numbers. Wortmannin-mediated PI3K/Akt pathway inhibition leads to increased osteoblastic differentiation at both low and high oxygen tension. We demonstrate that low oxygen stimulates a complex signaling network involving PI3K/Akt, Notch, and canonical Wnt pathways, which mediate the observed increase in nuclear Oct4a and REST, with simultaneous decrease in p53, AIF, and Bak. Collectively, these pathway activations contribute to increased self-renewal with concomitant decreased differentiation, cell cycle arrest, apoptosis, and/or senescence in MIAMI cells. Importantly, the PI3K/Akt pathway plays a central mechanistic role in the oxygen tension-regulated self-renewal versus osteoblastic differentiation of progenitor cells.

EGFR siRNA lipid nanocapsules efficiently transfect glioma cells in vitro.

Glioma are the most common malignant tumors of the central nervous system and remain associated with poor prognosis, despite the combination of chemotherapy and radiotherapy. EGFR targeting represents an interesting strategy to treat glioma. Indeed, a high level of endothelial growth factor receptors expression (EGFR), involved in the malignancy of the tumor, has been observed in glioma. Our strategy consisted in using EGFR siRNA entrapped into lipid nanocapsules (LNCs) via cationic liposomes. In vitro analyses on U87MG human glioma cells were performed to evaluate firstly the capacity of LNCs to efficiently deliver the siRNA and secondly the effect of EGFR siRNA targeting on U87MG proliferation. Then, the complement protein consumption was evaluated by CH50 assays to verify the suitability of the siRNA LNCs for systemic administration. The EGFR siRNA LNCs exhibited an adequate size lower than 150 nm as well as a neutral surface charge. The IC50 profile together with the 63% of protein extinction demonstrated the significant action of EGFR siRNA LNCs compared to scrambled LNCs. Dose and time-dependent survival assays showed a decrease of U87MG growth evaluated at 38%. Finally, low complement consumption demonstrated the suitability of EGFR siRNA LNCs for intravenous injection. In conclusion, EGFR siRNA LNCs demonstrated their capacity to efficiently encapsulate and deliver siRNA into U87MG human glioma cells, and will therefore be usable in the future for in vivo evaluation.

The therapeutic potential of human multipotent mesenchymal stromal cells combined with pharmacologically active microcarriers transplanted in hemi-parkinsonian rats.

Multipotent mesenchymal stromal cells (MSCs) raise great interest for brain cell therapy due to their ease of isolation from bone marrow, their immunomodulatory and tissue repair capacities, their ability to differentiate into neuronal-like cells and to secrete a variety of growth factors and chemokines. In this study, we assessed the effects of a subpopulation of human MSCs, the marrow-isolated adult multilineage inducible (MIAMI) cells, combined with pharmacologically active microcarriers (PAMs) in a rat model of Parkinson's disease (PD). PAMs are biodegradable and non-cytotoxic poly(lactic-co-glycolic acid) microspheres, coated by a biomimetic surface and releasing a therapeutic protein, which acts on the cells conveyed on their surface and on their microenvironment. In this study, PAMs were coated with laminin and designed to release neurotrophin 3 (NT3), which stimulate the neuronal-like differentiation of MIAMI cells and promote neuronal survival. After adhesion of dopaminergic-induced (DI)-MIAMI cells to PAMs in vitro, the complexes were grafted in the partially dopaminergic-deafferented striatum of rats which led to a strong reduction of the amphetamine-induced rotational behavior together with the protection/repair of the nigrostriatal pathway. These effects were correlated with the increased survival of DI-MIAMI cells that secreted a wide range of growth factors and chemokines. Moreover, the observed increased expression of tyrosine hydroxylase by cells transplanted with PAMs may contribute to this functional recovery.

Adult cell therapy for brain neuronal damages and the role of tissue engineering.

No long term effective treatments are currently available for brain neurological disorders such as stroke/cerebral ischemia, traumatic brain injury and neurodegenerative disorders. Cell therapy is a promising strategy, although alternatives to embryonic/foetal cells are required to overcome ethical, tissue availability and graft rejection concerns. Adult cells may be easily isolated from the patient body, therefore permitting autologous grafts to be performed. Here, we describe the use of adult neural stem cells, adrenal chromaffin cells and retinal pigment epithelium cells for brain therapy, with a special emphasis on mesenchymal stromal cells. However, major problems like cell survival, control of differentiation and engraftment remain and may be overcome using a tissue engineering strategy, which provides a 3D support to grafted cells improving their survival. New developments, such as the biomimetic approach which combines the use of scaffolds with extracellular matrix molecules, may improve the control of cell proliferation, survival, migration, differentiation and engraftment in vivo. Therefore, we later discuss scaffold properties required for brain cell therapy as well as new tissue engineering advances that may be implemented in combination with adult cells for brain therapy. Finally, we describe an approach developed in our laboratory to repair/protect lesioned tissues: the pharmacologically active microcarriers.

Mesenchymal and neural stem cells labeled with HEDP-coated SPIO nanoparticles: in vitro characterization and migration potential in rat brain.

Mesenchymal stem cells (MSC) may transdifferentiate into neural cells in vitro under the influence of matrix molecules and growth factors present in neurogenic niches. However, further experiments on the behavior of such stem cells remain to be done in vivo. In this study, rat MSC (rMSC) have been grafted in a neurogenic environment of the rat brain, the subventricular zone (SVZ), in order to detect and follow their migration using superparamagnetic iron oxide (SPIO) nanoparticles. We sought to characterize the potential effect of iron loading on the behavior of rMSC as well as to address the potential of rMSC to migrate when exposed to the adequate brain microenvironment. 1-hydroxyethylidene-1.1-bisphosphonic acid (HEDP)-coated SPIO nanoparticles efficiently labeled rMSC without significant adverse effects on cell viability and on the in vitro differentiation potential. In opposition to iron-labeled rat neural stem cells (rNSC), used as a positive control, iron-labeled rMSC did not respond to the SVZ microenvironment in vivo and did not migrate, unless a mechanical lesion of the olfactory bulb was performed. This confirmed the known potential of iron-labeled rMSC to migrate toward lesions and, as far as we know, this is the first study describing such a long distance migration from the SVZ toward the olfactory bulb through the rostral migratory stream (RMS).