Adipose‐derived stem cell spheroids are superior to single‐cell suspensions to improve fat autograft long‐term survival

Autologous fat transplantation is a widely used procedure for surgical reconstruction of tissues. The resorption rate of this transplantation remains high and unpredictable, reinforcing the need of adjuvant treatments that increase the long‐term stability of grafts. Adipose‐derived stem cells (ASC) introduced as single cells in fat has been shown clinically to reduce the resorption of fat grafts. On the other hand, the formulation of ASC into cell spheroids results in the enhancement of their regenerative potential. In this study, we developed a novel method to produce highly homogeneous ASC spheroids and characterized their features and efficacy on fat transplantation. Spheroids conserved ASC markers and multipotency. A regenerative gene expression profile was maintained, and genes linked to autophagy were upregulated whereas proliferation was decreased. Their secreted proteome was enriched in comparison with single‐cell ASC suspension. Addition of spheroids to fat graft in an animal model of transplantation resulted in a better graft long‐term stability when compared to single ASC suspension. In conclusion, we provide a novel method to manufacture homogenous ASC spheroids. These ASC spheroids are superior to ASC in single‐cell suspension to improve the stability of fat transplants, reinforcing their potential in reconstructive surgery.     

Adipose tissue‐derived progenitors for engineering osteogenic and vasculogenic grafts

The current need for bone grafts in orthopedic and reconstructive surgery cannot be satisfied by autologous tissue transplant due to its limited availability and significant associated morbidity. Tissue engineering approaches could supply sufficient amounts of bone substitutes by exploiting the ability to harvest autologous osteogenic progenitors associated with suitable porous materials. However, the generation of clinically relevant‐sized constructs is critically hampered by limited vascularization, with consequent engraftment and survival only of a thin outer shell, upon in vivo implantation. To overcome this limitation, different non‐mutually exclusive approaches have recently been developed to promote or accelerate graft vascularization, from angiogenic growth factor gene delivery to surgical pre‐vascularization of the construct before implantation. A simple, promising strategy involves the co‐culture of vasculogenic cells to form an intrinsic vascular network inside the graft in vitro, which can rapidly anastomose with the host blood vessels in vivo. Recent data have shown that adipose tissue‐derived stromal vascular fraction (SVF) may provide an efficient, convenient, and autologous source for both osteogenic and endothelial cells. When SVF progenitors were cultured in appropriate bioreactor systems and ectopically implanted, a functional vascular network connected to the host was formed concomitantly to bone formation. Future studies should aim at demonstrating that this approach effectively supports survival of scaled up cell‐based bone grafts at an orthotopic site. The procedure should also be adapted to become compatible with an intra‐operative timeline and complemented with the definition of suitable potency markers, to facilitate its development into a simplified, reproducible, and cost‐effective clinical treatment. J. Cell. Physiol. 225: 348–353, 2010. © 2010 Wiley‐Liss, Inc.     

Adipose stromal/stem cells assist fat transplantation reducing necrosis and increasing graft performance

Autologous fat transfer (AFT) is a procedure for adipose tissue (AT) repair after trauma, burns, post-tumor resections and lipodystrophies still negatively impacted by the lack of graft persistence. The reasons behind this poor outcome are unclear and seem to involve damages in either harvested/transplanted mature adipocytes or on their mesenchymal progenitors, namely adipose stromal/stem cells (ASC), and due to post-transplant AT apoptosis and involution. A rabbit subcutaneous AT regeneration model was here developed to first evaluate graft quality at different times after implant focusing on related parameters, such as necrosis and vasculogenesis. Standard AFT was compared with a strategy where purified autologous ASC, combined with hyaluronic acid (HA), assisted AFT. Five million of autologous ex vivo isolated CD29+, CD90+, CD49e+ ASC, loaded into HA, enriched 1 ml of AT generating an early significant protective effect in reducing AFT necrosis and increasing vasculogenesis with a preservation of transplanted AT architecture. This beneficial impact of ASC assisted AFT was then confirmed at three months with a robust lipopreservation and no signs of cellular transformation. By a novel ASC assisted AFT approach we ensure a reduction in early cell death favoring an enduring graft performance possibly for a more stable benefit in patients.     

The scope of mycoplasma contamination within the biopharmaceutical industry

Mycoplasma is well recognized as one of the most prevalent and serious microbial contaminants encountered within the manufacturing of biopharmaceuticals from the research phase to clinical development and production. The potential for mycoplasma contamination within cell culture systems was first identified by Robinson et al. in 1956 [1]. Presently, contamination rates in established cell cultures have been reported between 15 and 35% with considerably higher occurrence cited in certain selected populations [2]. In the last few years, there has been an expansion of diagnostic approaches for mycoplasma detection with the development and validation of rapid microbiological methods. The objective of this study was to determine current levels of mycoplasma infection of cell cultures, cell substrates and biologicals within a client based population. Retrospective comparison of 40,000 sample results was done to determine total contaminations rates amongst four (4) individual analytical assays. The establishment of reference data, such as existing contamination rates, becomes important in the critical appraisal of rapid microbiological methods for the detection of mycoplasma.     

Comparison of bacteriological qualities of various egg yolk sources and the in vitro and in vivo fertilizing potential of bovine semen frozen in egg yolk or lecithin based diluents

The addition of components of animal origin (egg yolk, milk) to most commercial diluents used to freeze bull semen represents a potential risk of contamination of the doses with bacteria or mycoplasma. A series of quantitative and qualitative analyses were performed to detect microbiological contamination observed in Biociphos plus (a new lecithin-glycerol based freezing salt buffer), in an egg yolk diluent (Triladyl) or in an egg yolk + milk-based (Laiciphos) diluent of bull semen. The 2 diluents containing animal products showed moderate (10 to 60 CFU/mL) contamination (17/17 samples) with bacteria or mycoplasma, or both, while no contamination was observed in the 6 examined batches of Biociphos plus. Biociphos plus was also compared with another commercial diluent (Laiciphos) for use in freezing bull semen intended for in vitro and/or in vivo fertilization. No difference (P > 0.05) could be detected between the 2 diluents for in vitro fertility rates (percentage of cleaved zygotes: 85.7% and 88.0%, respectively, for Laiciphos and Biociphos plus). Similarly, 2 series of comparisons conducted in dairy cows artificially inseminated with semen frozen in either Biociphos plus or Laiciphos showed no difference in fertilizing capacity (tested at 60 to 90 d; P > 0.05) irrespective of the age of the bulls (Trial 1, bulls aged 14 to 15 m.o.; Trial 2, bulls aged 2 to 5 yr, field trials). It is concluded that, in addition to maintaining the fertilizing capacity of bull semen at levels comparable to those observed with standard freezing diluent, Biociphos plus also prevents microbiological contamination by bacteria or mycoplasma, both of which are generally present in the various commercially available sources of egg yolk.     

Detection of mycoplasma contamination in cultured human fibroblasts : Comparison of biochemical and microbiological techniques

Eighteen human fibroblast strains were tested for mycoplasma contamination by polyacrylamide gel electrophoresis of ³H-uridine labeled RNA and by standard microbiological culture techniques. Despite negative culture results, prominent 23S and 16S RNA peaks were detected in 11 of these cell strains. The mycoplasma origin of this RNA was indicated by electron microscopic demonstration of these organisms. Another indication of contamination was the decreased specific radioactivity of whole cell RNA extracted from cell strains infected with mycoplasma.     

Albumin-coated structural lyophilized bone allografts: a clinical report of 10 cases

Bone replacement and the use of bone supplementary biological substances have become widespread in clinical practice. Although autografts have excellent properties, their limited availability, difficulties with shaping and donor site morbidity have made allografts a viable and increasingly preferred alternative. The main drawback of allografts is that the preparation destroys osteogenic cells and results in denaturation of osteoinductive proteins. Serum albumin is a well-known constituent of stem cell culture media and we found that lyophilizing albumin onto bone allografts markedly improves stem-cell attachment and bone healing in animal models thus replacing some of the osteoinductive potential. As a first step in the clinical introduction of albumin coated grafts, we aimed to test surgical handling and early incorporation in aseptic revision arthroplasty in humans. We selected patients who needed large structural allografts and the current operation was the last attempt at preserving a moving joint. In a series of 10 cases of hip and knee revision surgery we did not experience any drawbacks of the albumin-coated grafts during handling and implantation. Twelve months radiographic and SPECT-CT follow-up showed that the graft was well received by the host and active remodelling was observed. The lack of graft-related complications and the good 1-year results indicate that controlled trials may be initiated in more common bone grafting indications where long-term effectiveness can be evaluated.     

Adipose tissue-derived mesenchymal stem cell yield and growth characteristics are affected by the tissue-harvesting procedure

BACKGROUND: Adipose tissue contains a stromal vascular fraction that can be easily isolated and provides a rich source of adipose tissue-derived mesenchymal stem cells (ASC). These ASC are a potential source of cells for tissue engineering. We studied whether the yield and growth characteristics of ASC were affected by the type of surgical procedure used for adipose tissue harvesting, i.e. resection, tumescent liposuction and ultrasound-assisted liposuction. METHODS: Frequencies of ASC in the stromal vascular fraction were assessed in limiting dilution assays. The phenotypical marker profile of ASC was determined, using flow cytometry, and growth kinetics were investigated in culture. ASC were cultured under chondrogenic and osteogenic conditions to confirm their differentiation potential. RESULTS: The number of viable cells in the stromal vascular fraction was affected by neither the type of surgical procedure nor the anatomical site of the body from where the adipose tissue was harvested. After all three surgical procedures, cultured ASC did express a CD34+ CD31- CD105+ CD166+ CD45- CD90+ ASC phenotype. However, ultrasound-assisted liposuction resulted in a lower frequency of proliferating ASC, as well as a longer population doubling time of ASC, compared with resection. ASC demonstrated chondrogenic and osteogenic differentiation potential. DISCUSSION: We conclude that yield and growth characteristics of ASC are affected by the type of surgical procedure used for adipose tissue harvesting. Resection and tumescent liposuction seem to be preferable above ultrasound-assisted liposuction for tissue-engineering purposes.     

Adipose-derived stem cells alleviate osteoporosis by enchancing osteogenesis and inhibiting adipogenesis in a rabbit model

Background aimsOsteoporosis (OP) is characterized by a reduction in bone quality, which is associated with inadequacies in bone marrow mesenchymal stromal cells (BMSCs). As an alternative cell source to BMSCs, adipose-derived stem cells (ASCs) have been investigated for bone repair because of their osteogenic potential and self-renewal capability. Nevertheless, whether autologous ASCs can be used to promote bone regeneration under osteoporotic conditions has not been elucidated.MethodsThe OP rabbit model was established by means of bilateral ovariectomy (OVX). Both BMSCs and ASCs were harvested from OVX rabbits and expanded in vitro. The effects of osteogenic-induced ASCs on the in vitro adipogenic and osteogenic capabilities of BMSCs were evaluated. Autologous ASCs were then encapsulated by calcium alginate gel and transplanted into the distal femurs of OVX rabbits (n = 12). Hydrogel without loading cells was injected into the contralateral femurs as a control. Animals were killed for investigation at 12 weeks after transplantation.ResultsOsteogenic-induced ASCs were able to promote osteogenesis and inhibit adipogenesis of osteoporotic BMSCs through activation of the bone morphogenetic protein 2/bone morphogenetic protein receptor type IB signal pathway. Local bone mineral density began to increase at 8 weeks after ASC transplantation (P < 0.05). At 12 weeks, micro–computed tomography and histological evaluation revealed more new bone formation in the cell-treated femurs than in the control group (P < 0.05).ConclusionsThis study demonstrated that ASCs could stimulate proliferation and osteogenic differentiation of BMSCs in vitro and enhance bone regeneration in vivo, which suggests that autologous osteogenic-induced ASCs might be useful to alleviate OP temporally.     

Effect of tissue-harvesting site on yield of stem cells derived from adipose tissue: implications for cell-based therapies

The stromal vascular fraction (SVF) of adipose tissue contains an abundant population of multipotent adipose-tissue-derived stem cells (ASCs) that possess the capacity to differentiate into cells of the mesodermal lineage in vitro. For cell-based therapies, an advantageous approach would be to harvest these SVF cells and give them back to the patient within a single surgical procedure, thereby avoiding lengthy and costly in vitro culturing steps. However, this requires SVF-isolates to contain sufficient ASCs capable of differentiating into the desired cell lineage. We have investigated whether the yield and function of ASCs are affected by the anatomical sites most frequently used for harvesting adipose tissue: the abdomen and hip/thigh region. The frequency of ASCs in the SVF of adipose tissue from the abdomen and hip/thigh region was determined in limiting dilution and colony-forming unit (CFU) assays. The capacity of these ASCs to differentiate into the chondrogenic and osteogenic pathways was investigated by quantitative real-time polymerase chain reaction and (immuno)histochemistry. A significant difference (P = 0.0009) was seen in ASC frequency but not in the absolute number of nucleated cells between adipose tissue harvested from the abdomen (5.1 ± 1.1%, mean ± SEM) and hip/thigh region (1.2 ± 0.7%). However, within the CFUs derived from both tissues, the frequency of CFUs having osteogenic differentiation potential was the same. When cultured, homogeneous cell populations were obtained with similar growth kinetics and phenotype. No differences were detected in differentiation capacity between ASCs from both tissue-harvesting sites. We conclude that the yield of ASCs, but not the total amount of nucleated cells per volume or the ASC proliferation and differentiation capacities, are dependent on the tissue-harvesting site. The abdomen seems to be preferable to the hip/thigh region for harvesting adipose tissue, in particular when considering SVF cells for stem-cell-based therapies in one-step surgical procedures for skeletal tissue engineering.     

Repair of long-bone pseudoarthrosis with autologous bone marrow mononuclear cells combined with allogenic bone graft

Background aimsLong-bone pseudoarthrosis is a major orthopedic concern because of numerous factors such as difficulty of the treatment, high recurrence, high costs and the devastating effects on the patients' quality of life, which sometimes ends in amputation. Although the “gold standard” for the treatment of this pathology is autologous bone grafting, which has high osteogenic, osteoconductive and osteoinductive properties, this treatment presents some restrictions such as the limited amount of bone that can be taken from the patient and donor site morbidity. Bone marrow mononuclear cells (BM-MNCs) comprise progenitor and stem cells with pro-angiogenic and pro-osteogenic properties. Allogenic cancellous bone graft is a natural and biodegradable osteoconductive and osteoinductive scaffold. Combination of these two components could mimic the advantages of autologous bone grafting while avoiding its main limitations.MethodsLong-bone pseudoarthrosis was treated in seven patients with autologous BM-MNCs from iliac crest combined with frozen allogenic cancellous bone graft obtained from the tissue bank.ResultsAll patients showed complete bone consolidation 5.3 ± 0.9 months (range, 2–9 months) after cell transplantation. Moreover, limb pain disappeared in all of them. The mean follow-up was 35.8 ± 4.6 months after transplantation (range, 24–51 months) without pseudoarthrosis recurrence or pain reappearing.ConclusionsCombination of autologous BM-MNCs and allogenic bone graft could constitute an easy, safe, inexpensive and efficacious attempt to treat long-bone pseudoarthrosis and non-union by reproducing the beneficial properties of autologous bone grafting while restricting its disadvantages.     

Promoting long-term survival of insulin-producing cell grafts that differentiate from adipose tissue-derived stem cells to cure type 1 diabetes

Background

Insulin-producing cell clusters (IPCCs) have recently been generated in vitro from adipose tissue-derived stem cells (ASCs) to circumvent islet shortage. However, it is unknown how long they can survive upon transplantation, whether they are eventually rejected by recipients, and how their long-term survival can be induced to permanently cure type 1 diabetes. IPCC graft survival is critical for their clinical application and this issue must be systematically addressed prior to their in-depth clinical trials.

Methodology/Principal Findings

Here we found that IPCC grafts that differentiated from murine ASCs in vitro, unlike their freshly isolated islet counterparts, did not survive long-term in syngeneic mice, suggesting that ASC-derived IPCCs have intrinsic survival disadvantage over freshly isolated islets. Indeed, β cells retrieved from IPCC syngrafts underwent faster apoptosis than their islet counterparts. However, blocking both Fas and TNF receptor death pathways inhibited their apoptosis and restored their long-term survival in syngeneic recipients. Furthermore, blocking CD40-CD154 costimulation and Fas/TNF signaling induced long-term IPCC allograft survival in overwhelming majority of recipients. Importantly, Fas-deficient IPCC allografts exhibited certain immune privilege and enjoyed long-term survival in diabetic NOD mice in the presence of CD28/CD40 joint blockade while their islet counterparts failed to do so.

Conclusions/Significance

Long-term survival of ASC-derived IPCC syngeneic grafts requires blocking Fas and TNF death pathways, whereas blocking both death pathways and CD28/CD40 costimulation is needed for long-term IPCC allograft survival in diabetic NOD mice. Our studies have important clinical implications for treating type 1 diabetes via ASC-derived IPCC transplantation.     

Rapid detection of mycoplasma contamination in cell cultures using sybr green-based real-time polymerase chain reaction

Summary We have developed a simple method for rapid detection of mycoplasma contamination in cell cultures using SYBR Green-based real-time polymerase chain reaction (PCR). To detect eight common contaminant mollicutes, including Mycoplasma (M. arginini, M. fermentans, M. orale, M. hyorhinis, M. hominis, M. salivarium, M. pirum) and Acholeplasma laidlawii, four primers were prepared based on the 23S rRNA regions. Using these primers and a minimum of 100 fg of mycoplasma genomic DNA, the 23S rRNA regions of these eight mycoplasma species were consistently amplified by real-time PCR. In contrast, no specific specific amplification product was observed using DNA templates prepared from various mammalian cell lines. Frozen and cultured samples of several cell lines were tested for mycoplasma contamination to evaluated the utility of this method. Of 25 samples that tested positive for mycoplasma by Hoechst staining, which requires two passages of cell cultures started from frozen samples, mycoplasma was detected by real-time PCR in 24 samples of cell extracts prepared directly from frozen samples. When cultured samples were used for this assay, the accuracy of the diagnoses was further improved. Thus, this technique, which is simple, rapid, and sensitive enough for practical application, in suitable for handling many samples and for routine screening for mycoplasma contamination of cell cultures.     

Curative therapy for hemoglobinopathies: an International Society for Cell & Gene Therapy Stem Cell Engineering Committee review comparing outcomes,accessibility and cost of ex vivo stem cell gene therapy versus allogeneic hematopoietic stem cell transplantation

Thalassemia and sickle cell disease (SCD) are the most common monogenic diseases in the world and represent a growing global health burden. Management is limited by a paucity of disease-modifying therapies; however, allogeneic hematopoietic stem cell transplantation (HSCT) and autologous HSCT after genetic modification offer patients a curative option. Allogeneic HSCT is limited by donor selection, morbidity and mortality from transplant conditioning, graft-versus-host disease and graft rejection, whereas significant concerns regarding long-term safety, efficacy and cost limit the broad applicability of gene therapy. Here the authors review current outcomes in allogeneic and autologous HSCT for transfusion-dependent thalassemia and SCD and provide our perspective on issues surrounding accessibility and costs as barriers to offering curative therapy to patients with hereditary hemoglobinopathies.     

Mycoplasma contamination of cell cultures: methods for its detection and the possible routes of Mycoplasma infection spread

83 continuous cell lines were screened for mycoplasma contamination by three methods: microbiological seeding for enriched media, staining with Hoechst-33258 stain, and autoradiography with 3H-thymidine incorporation. It has been shown that combination of different methods is necessary for the strict control of mycoplasma-contamination in cell cultures. Simultaneous manipulation with mycoplasma infected and pure cell lines leads to cross-contamination in 2-3 passages. Precautions are described to preclude mycoplasma-contamination during prolonged cell cultivation.     

Human polymer-based cartilage grafts for the regeneration of articular cartilage defects

The use of autologous chondrocyte implantation (ACI) and its further development combining autologous chondrocytes with bioresorbable matrices may represent a promising new technology for cartilage regeneration in orthopaedic research. Aim of our study was to evaluate the applicability of a resorbable three-dimensional polymer of pure polyglycolic acid (PGA) for the use in human cartilage tissue engineering under autologous conditions. Adult human chondrocytes were expanded in vitro using human serum and were rearranged three-dimensionally in human fibrin and PGA. The capacity of dedifferentiated chondrocytes to re-differentiate was evaluated after two weeks of tissue culture in vitro and after subcutaneous transplantation into nude mice by propidium iodide/fluorescein diacetate (PI/FDA) staining, scanning electron microscopy (SEM), gene expression analysis of typical chondrocyte marker genes and histological staining of proteoglycans and type II collagen. PI/FDA staining and SEM documented that vital human chondrocytes are evenly distributed within the polymer-based cartilage tissue engineering graft. The induction of the typical chondrocyte marker genes including cartilage oligomeric matrix protein (COMP) and cartilage link protein after two weeks of tissue culture indicates the initiation of chondrocyte re-differentiation by three-dimensional assembly in fibrin and PGA. Histological analysis of human cartilage tissue engineering grafts after 6 weeks of subcutaneous transplantation demonstrates the development of the graft towards hyaline cartilage with formation of a cartilaginous matrix comprising type II collagen and proteoglycan. These results suggest that human polymer-based cartilage tissue engineering grafts made of human chondrocytes, human fibrin and PGA are clinically suited for the regeneration of articular cartilage defects.     

Optimized PCR-based Detection of Mycoplasma

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. Although mycoplasma do not usually kill contaminated cells, they are difficult to detect and can cause a variety of effects on cultured cells, including altered metabolism, slowed proliferation and chromosomal aberrations. In short, mycoplasma contamination compromises the value of those cell lines in providing accurate data for life science research.The sources of mycoplasma contamination in the laboratory are very challenging to completely control. As certain mycoplasma species are found on human skin, they can be introduced through poor aseptic technique. Additionally, they can come from contaminated supplements such as fetal bovine serum, and most importantly from other contaminated cell cultures. Once mycoplasma contaminates a culture, it can quickly spread to contaminate other areas of the lab. Strict adherence to good laboratory practices such as good aseptic technique are key, and routine testing for mycoplasma is highly recommended for successful control of mycoplasma contamination.PCR-based detection of mycoplasma has become a very popular method for routine cell line maintenance. PCR-based detection methods are highly sensitive and can provide rapid results, which allows researchers to respond quickly to isolate and eliminate contamination once it is detected in comparison to the time required using microbiological techniques. The LookOut Mycoplasma PCR Detection Kit is highly sensitive, with a detection limit of only 2 genomes per μl. Taking advantage of the highly specific JumpStart Taq DNA Polymerase and a proprietary primer design, false positives are greatly reduced. The convenient 8-tube format, strips pre-coated with dNTPs, and associated primers helps increase the throughput to meet the needs of customers with larger collections of cell lines.Given the extreme sensitivity of the kit, great care must be taken to prevent inadvertent contamination of samples and reagents. The step-by-step protocol we demonstrate highlights the precautions and practices required for reliable mycoplasma detection. We also show and discuss typical results and their interpretation. Our goal is to ensure the success of researchers using the LookOut Mycoplasma PCR Detection Kit.Download video file.^{(36M, mov)}     

Interleukin (IL)-10 induced by CD11b(+) cells and IL-10-activated regulatory T cells play a role in immune modulation of mesenchymal stem cells in rat islet allografts

Mesenchymal stem cells (MSCs) are suggested to be immune modulators because of their therapeutic potential in transplantation. In the present study, we evaluated the therapeutic potential of autologous MSCs for preventing graft rejection after allogeneic rat islet transplantation. We assessed the ability of MSCs to elicit an antiproliferative response in alloreactive lymphocytes and tested the immunosuppressive effect of MSCs in allogeneic islet transplantation. In islet allotransplantation, injection of autologous MSCs or a subtherapeutic dose of cyclosporine A (CsA; 5 mg/kg) alone did not prolong allograft survival. However, graft survival was attained for >100 d in 33% of autologous MSC-plus-CsA-treated recipients, indicating that graft acceptance was achieved in a subgroup of allograft recipients. Splenocytes from autologous MSC-plus-CsA-treated rats exhibited a reduced mixed lymphocyte reaction (MLR)-proliferative response to donor stimulators and increased interleukin (IL)-10 release. Interestingly, after excluding host CD11b(+) cells, splenic T cells from autologous MSC-plus-CsA-treated rats did not produce IL-10 or did not inhibit proliferative responses under the same conditions. The use of autologous MSC-plus-CsA downregulated immune responses, inducing donor-specific T-cell hyporesponsiveness by reducing the production of proinflammatory cytokines and inducing antiinflammatory cytokine production, especially that of IL-10, during the early posttransplantation period. T-regulatory cells made a contribution at a later phase. In conclusion, the combined use of autologous MSCs and low-dose CsA exerted a synergistic immunosuppressive effect in an islet allograft model, suggesting a role for autologous MSCs as an immune modulator.     

Posterolateral spinal fusion with ostegenesis induced BMSC seeded TCP/HA in a sheep model

Autogenous bone graft is the gold standard for fusion procedure. However, pain at donor site and inconsistent outcome have left a surgeon to venture into some other technique for spinal fusion. The objective of this study was to determine whether osteogenesis induced bone marrow stem cells with the combination of ceramics granules (HA or TCP/HA), and fibrin could serve as an alternative to generate spinal fusion. The sheep's bone marrow mesenchymal stem cells (BMSCs) were aspirated form iliac crest and cultured for several passages until confluence. BMSCs were trypsinized and seeded on hydroxyapatite scaffold (HA) and tricalcium phosphate/hydroxyapatite (TCP/HA) for further osteogenic differentiation in the osteogenic medium one week before implantation. Six adult sheep underwent three-level, bilateral, posterolateral intertransverse process fusions at L1–L6. Three fusion sites in each animal were assigned to three treatments: (a) HA constructs group/L1–L2, (b) TCP/HA constructs group/L2–L3, and (c) autogenous bone graft group/L5–L6. The spinal fusion segments were evaluated using radiography, manual palpation, histological analysis and scanning electron microscopy (SEM) 12 weeks post implantation. The TCP/HA constructs achieved superior lumbar intertransverse fusion compared to HA construct but autogenous bone graft still produced the best fusion among all.