Bone marrow-derived mesenchymal stromal cells accelerate wound healing in the rat

Bone marrow-derived mesenchymal stromal cells (BMSCs) are multipotential stem cells capable of differentiation into numerous cell types, including fibroblasts, cartilage, bone, muscle, and brain cells. BMSCs also secrete a large number of growth factors and cytokines that are critical to the repair of injured tissues. Because of the extraordinary plasticity and the ability of syngeneic or allogeneic BMSCs to secrete tissue-repair factors, we investigated the therapeutic efficacy of BMSCs for healing of fascial and cutaneous incisional wounds in Sprague-Dawley rats. Systemic administration of syngeneic BMSCs (2 x 10(6)) once daily for 4 days or a single treatment with 5 x 10(6) BMSCs 24 hours after wounding significantly increased the wound bursting strength of fascial and cutaneous wounds on days 7 and 14 postwounding. Wound healing was also significantly improved following injection of BMSCs locally at the wound site. Furthermore, allogeneic BMSCs were as efficient as syngeneic BMSCs in promoting wound healing. Administration of BMSCs labeled with iron oxides/1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate fluorescent dye revealed that systemically administered BMSCs engraft to the wound. The increase in the tensile strength of wounds treated with BMSCs was associated with increased production of collagen in the wound. In addition, BMSC treatment caused more rapid histologic maturation of wounds compared with untreated wounds. These data suggest that cell therapy with BMSCs has the potential to augment healing of surgical and cutaneous wounds.     

Challenging abdominal incisional hernia repaired with platelet-rich plasma and bone marrow-derived mesenchymal stromal cells. A case report

IntroductionThe necessity to develop new treatment options for challenging procedures in hernia surgery is becoming even more evident and tissue engineering and biological technologies offer even newer strategies to improve fascial healing. The present case reports a patient-tailored surgical technique performed to repair a grade IV abdominal incisional hernia, with a combined use of platelet-rich plasma and bone marrow-derived mesenchymal stromal cells, implanted on a biological mesh.Presentation of the caseA 71 year-old female patient complained of an abdominal incisional hernia, complicated by enterocutaneous fistula, four-months following laparostomy. Contrast enhanced computed tomography showed an incisional hernia defect of 15.5 × 20 cm, with a subcutaneous abscess and an intestinal loop adherent to the anterior abdominal wall, with a concomitant enterocutaneous fistula. Surgery involved abdominal wall standardized technique closure, with in addition platelet-rich plasma and bone marrow-derived mesenchymal stromal cells implanted on a biological mesh. Two years follow up showed no recurrences of incisional hernia.DiscussionCoating surgical meshes with patient’s own cells may improve biocompatibility, by reducing inflammation and adhesion formation. Moreover, platelet-rich plasma is a good source of growth factors for wound healing, as well as a good medium for bone marrow multinucleate cells introduction into fascial repair.ConclusionThis approach is likely to improve abdominal wall repair in high grade (IV) incisional hernia, with the real possibility of improving prosthetic compatibility and reducing future recurrences. The authors agree with the necessity of further studies and trials to assure the safety profile and superiority of this procedure.     

Transplantation of marrow-derived mesenchymal stem cells and platelet-rich plasma during distraction osteogenesis--a preliminary result of three cases

Clinical results of distraction osteogenesis with transplantation of marrow-derived mesenchymal stem cells (MSCs) and platelet-rich plasma (PRP) were reviewed in three femora and two tibiae of the two patients with achondroplasia and one patient with congenital pseudarthrosis of the tibia. MSCs derived from the iliac crest were cultured with osteogenic supplements and differentiated into osteoblast-like cells. PRP, which is known to contain several growth factors and coagulate immediately by a minute introduction of thrombin and calcium, was prepared just before transplantation. Culture-expanded osteoblast-like cells and autologous PRP were injected into the distracted callus with the thrombin-calcium mixture so that the PRP gel might develop within the injected site. Transplantation of MSCs and PRP was done at the lengthening and consolidation period in each patient. The target lengths were obtained in every leg without major complications and the average healing index was 23.0 days/cm (18.8-26.9 days/cm). Although these results are still preliminary, transplantation of osteoblast-like cells and PRP, which seemed to be a safe and minimally invasive cell therapy, could shorten the treatment period by acceleration of bone regeneration during distraction osteogenesis.     

骨髓间充质干细胞对同种异体胰岛的保护作用

目的 研究小鼠骨髓间充质干细胞(mesenehymal stem cells,MSCs)对同种异体胰岛的保护作用.方法 将C57BL/6小鼠骨髓间充质干细胞按照3×104/孔的密度预先接种至24孔培养板,次日分离纯化BALB/c小鼠胰岛,将其分为链脲菌素(streptozotocin,STZ)处理(诱导化学损伤)、混合淋巴细胞反应(mixed lymphocyte reaction,MLR)处理(诱导免疫损伤)、空白处理三组,与预先接种的MSCs进行共培养.作为实验对照,另设三组胰岛在不加MSCs的条件下进行体外培养.5 d后,用吖啶橙(acridine orange,AO)/碘化丙啶(propidium iodide,PI)荧光染色评估胰岛活力,葡萄糖刺激的胰岛素分泌实验检测其功能,比较MSCs共培养组与对照组胰岛在低糖、高糖刺激下的胰岛素分泌量及刺激指数(即高糖刺激胰岛素分泌量/低糖刺激胰岛素分泌量比值).结果 AO/PI染色显示,STZ或MLR处理的胰岛中有大量红色荧光的死细胞,而与MSCs共培养的胰岛中死细胞明显减少,绿色荧光的活细胞明显增加;STZ或MLR处理组胰岛在低糖、高糖刺激下的胰岛素分泌水平及刺激指数均显著下降,而与MSCs共培养者较相应对照组胰岛功能明显改善(P＜0.05).结论 小鼠骨髓间充质干细胞可减轻同种异体胰岛的化学及免疫损伤,保护游离胰岛的活力与功能.

Abstract：

Objective To study the protection of mouse bone marrow-derived mesenchymal stem cells ( MSCs) for allogenic islets. Method MSCs from C57BL/6 mice were preceded to a 24-well culture plate with the density of 3 × 104/well. On the second day, islets were isolated, purified and divided to undergo streptozotocin (STZ) induced chemical injury and mixed lymphocyte reaction ( MLR) respectively. Then, treated and control islets were respectively divided into the following groups: islets + MSCs, STZ-islets + MSCs, and MLR-islets + MSCs. As control groups for their counterparts, treated or non-treated islets were also cultured without MSCs. On the 5th day incubation, glucose-stimulated insulin secretion test was performed to assess the function of islets in different groups, comparing their insulin-secretion amount stimulated by low or high glucose and the stimulation index determined by the ratio of (insulin amount secreted under high-glucose stimulation)/(insulin amount secreted under low-glucose stimulation ). Islet viability was evaluated by acridine orange (AO)/propidium iodide (PI) fluorescence staining. Result As shown by AO/PI staining, large numbers of dead cell with red fluorescence could be observed in STZ- or MLR- treated islets without MSCs, while the number of dead cells obviously reduced in MSC-cocultured islets with increased viable cells of green fluorescence. STZ- or MLR- treated islets exhibited apparently decreased insulin-secretion amount either under low- or high-glucose stimulation, as well as the stimulation index. The insulin-secretion function was significantly improved in islets cocultured with allogenic MSCs (P ＜ 0. 05 ). Conclusions Bone marrow-derived MSCs can protect isolated allogenic islets against chemical and immunological injury.     

Placental mesenchymal stromal cells seeded on clinical grade extracellular matrix improve ambulation in ovine myelomeningocele

Purpose

The purpose of this study was to investigate the effects of placental mesenchymal stromal cells (PMSCs) seeded on a clinical grade porcine small intestinal submucosa (SIS)-derived extracellular matrix (ECM) on hindlimb motor function in an ovine fetal repair model of myelomeningocele (MMC).

Stromal stem cells and platelet-rich plasma improve bone allograft integration

Early vascular invasion is a key factor in bone allograft incorporation. It may reduce the complications related to slow and incomplete bone integration. Bone-marrow-derived stromal stem cells associated with platelet-rich plasma are potent angiogenic inducers proven to release vascular endothelial growth factor. Our goal was to test whether the combination of stromal stem cells and platelet-rich plasma is able to increase massive allograft integration in a large animal model with sacrifice at 4 months. A critical defect was made in the mid-diaphysis of the metatarsal bone of 10 sheep; the study group received an allograft plus stromal stem cells, platelet-rich plasma, and collagen (six animals) and the control group received only the allograft (four animals). Investigation was done with radiographs, mechanical tests and histomorphometric analysis, including new vascularization. Results showed substantial new bone formation in the allograft of the study group. Bone formation is correlated with better vascular invasion and remodeling of the graft in the study group. These results confirm the key role played by stromal stem cells and platelet-rich plasma in bone repair. Further studies are needed to better define the role stromal stem cells play when implanted alone.     

Synergy of tendon stem cells and platelet-rich plasma in tendon healing

Injured rat Achilles tendons were treated with botulism toxin to create a mechanically unloaded condition (unloaded) or left untreated (loaded), and then treated with phosphate‐buffered saline (PBS), platelet‐rich plasma (PRP), tendon stem cells (TSCs), or a combination (TSCs + PRP). mRNA and protein expression of collagen I, collagen III, tenascin C, and Smad 8 were determined by real time PCR and immunostaining, respectively. Loaded tendons treated with PBS, PRP, or TSCs for 3 or 14 days had higher collagen I mRNA expression than unloaded tendons. Loaded tendons treated with PBS for 3 or 14 days or with PRP for 3 days had higher collagen I protein levels than unloaded tendons. Loaded tendons treated for 3 days with PBS, for 14 days with PRP or TSCs or TSCs + PRP for 3 or 14 days had higher collagen III protein levels than unloaded tendons. Collagen I mRNA levels were higher in TSCs + PRP‐treated loaded tendons compared to PBS‐treated loaded tendons on day 3 of treatment. Based on changes in the expression of tendon‐healing genes, our data suggest that the combination of TSCs and PRP has synergistic effects on tendon healing under both loaded and unloaded conditions, and loaded conditions improve tendon healing. © 2011 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:991–997, 2012     

Hormesis mediates platelet-rich plasma and wound healing

Platelet-rich plasma (PRP) has become an accepted and general wound healing approach with an extremely wide range of applications. Despite considerable diversity in the composition of platelet-rich plasma products that are applied in specific wound healing usage, it is widely recognised that such diverse platelet-rich plasma complex mixtures routinely display hormetic-like biphasic concentrations that are independent of the tissue treated and endpoints measured. The present paper is the first to place the area of platelet-rich plasma-biomedical research and applications within an hormetic framework. The platelet-rich plasma area is also unique as it represents the application of the hormetic concept to the issue of complex biological mixtures.     

Treatment with bone marrow-derived stromal cells accelerates wound healing in diabetic rats

Bone marrow stem cells participate in tissue repair processes and may have a role in wound healing. Diabetes is characterised by delayed and poor wound healing. We investigated the potential of bone marrow-derived mesenchymal stromal cells (BMSCs) to promote healing of fascial wounds in diabetic rats. After manifestation of streptozotocin (STZ)-induced diabetic state for 5 weeks in male adult Sprague-Dawley rats, healing of fascial wounds was severely compromised. Compromised wound healing in diabetic rats was characterised by excessive polymorphonuclear cell infiltration, lack of granulation tissue formation, deficit of collagen and growth factor [transforming growth factor (TGF-beta), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor PDGF-BB and keratinocyte growth factor (KGF)] expression in the wound tissue and significant decrease in biomechanical strength of wounds. Treatment with BMSC systemically or locally at the wound site improved the wound-breaking strength (WBS) of fascial wounds. The improvement in WBS was associated with an immediate and significant increase in collagen levels (types I-V) in the wound bed. In addition, treatment with BMSCs increased the expression of growth factors critical to proper repair and regeneration of the damaged tissue moderately (TGF-beta, KGF) to markedly (EGF, VEGF, PDGF-BB). These data suggest that cell therapy with BMSCs has the potential to augment healing of the diabetic wounds.     

Bone marrow-derived cells in the healing burn wound--more than just inflammation

Scarring after severe burn is a result of changes in collagen deposition and fibroblast activity that result in repaired but not regenerated tissue. Re-epithelialisation of wounds and dermal cell repopulation has been thought to be driven by cells in the periphery of the wound. However, recent research demonstrated that cells originating from the bone marrow contribute to healing wounds in other tissues and also after incisional injury. We investigated the contribution of bone marrow-derived cells to long-term cell populations in scar tissue (primarily fibroblasts and keratinocytes) after severe burn. Wild-type mice were lethally irradiated and then the bone marrow reconstituted by injection of chimeric bone marrow cells expressing EGFP marker protein. Mice with chimeric bone marrow were then given a burn, either an 1-cm diameter injury (to mimic minor injury) or 2-cm diameter (to mimic moderate injury). Wounds were analysed at days 1, 3, 7, 14, 21, 28, 56 and 120 using FACS and immunohistochemistry to identify the percentage and cell type within the wound originating from the bone marrow. The inflammatory cell infiltrate at the early time-points was bone marrow in origin. At later time-points, we noted that over half of the fibroblast population was bone marrow-derived; we also observed that a small percentage of keratinocytes appeared to be bone marrow in origin. These findings support the theory that the bone marrow plays an important role in providing cells not only for inflammation but also dermal and epidermal cells during burn wound healing. This increases our understanding of cell origins in the healing wound, and has the potential to impact on clinical practice providing a potential mechanism for intervention away from conventional topical treatments and directed instead to systemic treatments affecting the bone marrow response.     

The platelet-rich plasma and mesenchymal stem cell milieu: A review of therapeutic effects on bone healing

Platelet-rich plasma is autologous plasma that contains concentrated platelets compared to whole blood. It is relatively inexpensive to produce, can be easily isolated from whole blood, and can be administered while the patient is in the operating room. Further, because platelet-rich plasma is an autologous therapy, there is minimal risk for adverse reactions to the patient. Platelet-rich plasma has been used to promote bone regeneration due to its abundance of concentrated growth factors that are essential to wound healing. In this review, we summarize the methods for producing platelet-rich plasma and the history of its use in bone regeneration. We also summarize the growth factor profiles derived from platelet-rich plasma, with emphasis on those factors that play a direct role in promoting bone repair within the local fracture environment. In addition, we discuss the potential advantages of combining platelet-rich plasma with mesenchymal stem cells, a multipotent cell type often obtained from bone marrow or fat, to improve craniofacial and long bone regeneration. We detail what is currently known about how platelet-rich plasma influences mesenchymal stem cells in vitro, and then highlight the clinical outcomes of administering platelet-rich plasma and mesenchymal stem cells as a combination therapy to promote bone regeneration in vivo.     

Bone marrow-derived mesenchymal stem cells influence early tendon-healing in a rabbit achilles tendon model

BACKGROUND: A repaired tendon needs to be protected for weeks until it has accrued enough strength to handle physiological loads. Tissue-engineering techniques have shown promise in the treatment of tendon and ligament defects. The present study tested the hypothesis that bone marrow-derived mesenchymal stem cells can accelerate tendon-healing after primary repair of a tendon injury in a rabbit model. METHODS: Fifty-seven New Zealand White rabbits were used as the experimental animals, and seven others were used as the source of bone marrow-derived mesenchymal stem cells. The injury model was a sharp complete transection through the midsubstance of the Achilles tendon. The transected tendon was immediately repaired with use of a modified Kessler suture and a running epitendinous suture. Both limbs were used, and each side was randomized to receive either bone marrow-derived mesenchymal stem cells in a fibrin carrier or fibrin carrier alone (control). Postoperatively, the rabbits were not immobilized. Specimens were harvested at one, three, six, and twelve weeks for analysis, which included evaluation of gross morphology (sixty-two specimens), cell tracing (twelve specimens), histological assessment (forty specimens), immunohistochemistry studies (thirty specimens), morphometric analysis (forty specimens), and mechanical testing (sixty-two specimens). RESULTS: There were no differences between the two groups with regard to the gross morphology of the tendons. The fibrin had degraded by three weeks. Cell tracing showed that labeled bone marrow-derived mesenchymal stem cells remained viable and present in the intratendinous region for at least six weeks, becoming more diffuse at later time-periods. At three weeks, collagen fibers appeared more organized and there were better morphometric nuclear parameters in the treatment group (p < 0.05). At six and twelve weeks, there were no differences between the groups with regard to morphometric nuclear parameters. Biomechanical testing showed improved modulus in the treatment group as compared with the control group at three weeks (p < 0.05) but not at subsequent time-periods. CONCLUSIONS: Intratendinous cell therapy with bone marrow-derived mesenchymal stem cells following primary tendon repair can improve histological and biomechanical parameters in the early stages of tendon-healing.     

骨髓间充质干细胞促进骨-肌腱结合部位早期愈合的动物生物力学实验研究

[目的]通过生物力学研究,观察骨髓间充质干细胞对骨-肌腱结合部愈合的影响.[方法]采用骨髓穿刺、全骨髓培养法获取兔骨髓间充质干细胞.24只18周龄新西兰大白兔随机分为2组,实验组将骨髓间充质干细胞与Pluronic F-127载体材料结合后,植入兔髌骨部分切除模型,对照组只进行手术,不植入细胞.在术后6、12、18周处死动物取标本(n=4)进行生物力学检测评估骨-肌腱结合部位的愈合恢复情况.[结果]生物力学结果显示相同时间点实验组拉断负荷及极限拉应力均大于对照组,差异有统计学意义(P<0.05),提示实验组恢复较对照组迅速.[结论]相同时间点实验组拉断负荷、极限拉应力均大于对照组.实验组的力学特性明显高于对照组.骨髓间充质干细胞可以促进骨-肌腱结合部细胞早期愈合,提高其力学特性.     

Systemically administered mesenchymal stromal cells transduced with insulin-like growth factor-I localize to a fracture site and potentiate healing

OBJECTIVE: To determine to the ability of systemically administered pluripotential mesenchymal stromal cells to localize to a fracture site and whether transduction with a therapeutic gene, insulin-like growth factor-I (D1-IGF-I), could potentiate healing. DESIGN: Murine model, basic science study. SETTING: Laboratory. SPECIMENS: Closed, transverse, mid-shaft femur fractures were produced in 108 Balb/c mice after intramedullary stabilization. INTERVENTIONS: A cloned, pluripotential, mesenchymal cell line, termed D1, was stably transfected with either the gene beta-galactosidase (D1-BAG) as a histologic marker or with the gene IGF-I (D1-IGF-I) growth factor. Mice received systemic injections of either D1-BAG cells for in vivo localization or D1-IGF-I for therapeutic intervention. A third group received lactated Ringer's solution and served as control. MAIN OUTCOME MEASUREMENTS: Sections obtained from the fracture site and contralateral femurs were examined histologically and by deoxyribonucleic acid-polymerase chain reaction (DNA-PCR) to detect the presence of transplanted cells at 2, 4, and 6 weeks after fracture. Matrix mineralization and callus maturation were evaluated by histology. RESULTS: At all time points, using histologic staining with X-gal and deoxyribonucleic acid-polymerase chain reaction for marker genes, there was a statistically greater number of transplanted cells ( p< 0.001) and significantly higher DNA-PCR for marker genes ( p< 0.001) in the fractured femurs than in the nonfractured femurs. Mice receiving D1-IGF-I cells demonstrated a greater percent of mineralized callus than controls at two weeks (p < 0.05). At 4 and 6 weeks, treated mice demonstrated on average greater mineralized matrix and accelerated progression to an osseous callus as compared with the control group. CONCLUSIONS: Cell-based gene therapy has the potential to deliver higher therapeutic levels of growth factors specifically at the site of cell localization while minimizing wider systemic side effects. This study demonstrates that systemically injected IGF-I transduced, mesenchymal cells are able to return to and repopulate the bone marrow. More importantly, these cells localize preferentially to a fracture site and accelerated fracture healing.