The role of bone marrow mononuclear cell-conditioned medium in the proliferation and migration of human dermal fibroblasts.

BACKGROUND: Several recent studies have demonstrated the great potential of bone marrow cells in regenerative medicine, not only for their ability to differentiate to match a damaged cell type, but also because they synthesize and release various growth factors and cytokines.We examined the effect of bone marrow cell-conditioned medium in the healing process, especially in terms of fibroblast proliferation and migration. METHODS: These in vitro studies consisted of co-culture (without direct contact) of dermal fibroblasts with mononuclear bone marrow cells and the use of conditioned medium obtained from these cultures in a scratch wound model. RESULTS: Mononuclear cells were found to increase the proliferation of fibroblasts, and the conditioned medium showed a stimulatory effect on the migration of fibroblasts. CONCLUSION: When considered together with the observed increase in growth factor levels in conditioned medium, it appears that these cells act through a paracrine mechanism.

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

BACKGROUND AIMS: Long-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. METHODS: Long-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. RESULTS: All 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. CONCLUSIONS: Combination 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.

Advances in stem cell therapy.

Since the beginning of stem cell biology, considerable effort has been focused in the translation of scientific insights into new therapies. Cell-based assays represent a new strategy for organ and tissue repair in several pathologies. Moreover, alternative treatment strategies are urgently needed due to donor organ shortage that costs many lives every year and results in lifelong immunosuppression. At the moment, only the use of hematopoietic stem cells is considered as the standard for the treatment of malignant and genetic bone marrow disorders, being all other stem cell applications highly experimental. The present chapter tries to summarize some ongoing approaches of stem cell regenerative medicine and also introduces recent findings from published studies and trials conducted in various tissues such as skeletal muscle, liver and lung.

Tissue bioengineering and artificial organs.

The scarcity of organs and tissues for transplant and the need of immunosuppressive drugs to avoid rejection constitute two reasons that justify organ and tissue production in the laboratory. Tissue engineering based tissues (TE) could allow to regenerate the whole organ from a fragment or even to produce several organs from an organ donor for grafting purposes. TE is based in: (1) the ex vivo expansion of cells, (2) the seeding of these expanded cells in tridimensional structures that mimic physiological conditions and, (3) grafting the prototype. In order to graft big structures it is necessary that the organ or tissue produced "ex vivo" bears a vascular tree to ensure the nutrition of its deep layers. At present, no technology has been developed to provide this vascular tree to TE derived products. Thus, these tissues must be thin enough to acquire nutrients during the first days by diffusion from surrounding tissues. This fact constitutes nowadays the greatest limitation of technologies for organ development in the laboratory.In this chapter, all these problems and their possible solutions are commented. Also, the present status of TE techniques in the regeneration of different organ systems is reviewed.

Regeneration of mandibular defects using adipose tissue mesenchymal stromal cells in combination with human serum-derived scaffolds.

Bone regeneration is a challenging issue. Traditional solutions bring risks, potential complications, and morbidity. The aim of the present study was to regenerate critical-sized mandible defects in athymic rats with adipose tissue mesenchymal stromal cells (AT-MSCs) in combination with human serum-derived scaffolds. Two approaches to treatment were performed. The first approach used differentiated stromal cells that became osteogenic cell lines. The second approach used no pre-differentiation. Follow-up periods were 45 days and 90 days. Both cell types were combined with human serum-derived scaffolds. Afterward, histological (haematoxylin-eosin and Masson's Trichrome stain modified by Goldner), immunohistochemical (human vimentin and Stro-1), and radiological (microCT) studies were performed. The level of calcification between the groups was compared by analysis of variance, and statistical significance was set at p < 0.05. The results demonstrate that bone regeneration can be achieved with both undifferentiated and pre-differentiated cells, but that the structure and level of calcification were better achieved with pre-differentiated cells (p < 0.05). The scaffold is suitable for this cell type, is osteoconductive and simple to perform. This article highlights the possible application of adipose tissue mesenchymal stromal cells in combination with a non-mineralized scaffold in bone regeneration.

CD271 as a marker to identify mesenchymal stem cells from diverse sources before culture.

Mesenchymal stem cells, due to their characteristics are ideal candidates for cellular therapy. Currently, in culture these cells are defined by their adherence to plastic, specific surface antigen expression and multipotent differentiation potential. However, the in vivo identification of mesenchymal stem cells, before culture, is not so well established. Pre-culture identification markers would ensure higher purity than that obtained with selection based on adherence to plastic. Up until now, CD271 has been described as the most specific marker for the characterization and purification of human bone marrow mesenchymal stem cells. This marker has been shown to be specifically expressed by these cells. Thus, CD271 has been proposed as a versatile marker to selectively isolated and expand multipotent mesenchymal stem cells with both immunosuppressive and lymphohematopoietic engraftment-promoting properties. This review focuses on this marker, specifically on identification of mesenchymal stem cells from different tissues. Literature revision suggests that CD271 should not be defined as a universal marker to identify mesenchymal stem cells before culture from different sources. In the case of bone marrow or adipose tissue, CD271 could be considered a quite suitable marker; however this marker seems to be inadequate for the isolation of mesenchymal stem cells from other tissues such as umbilical cord blood or wharton's jelly among others.

Brain death effects on catecholamine levels and subsequent cardiac damage assessed in organ donors.

BACKGROUND: Brain death (BD) causes hemodynamic and neuroendocrine alterations including a catecholamine surge, which in turn causes histologic lesions in cardiac muscle such as contraction bands, focal mononuclear cell infiltrates and cardiomyocyte necrosis. These changes are likely to compromise heart function and could therefore also affect the graft response after heart transplantation. This study was designed to examine the catecholamine surge, the catecholamine release pattern and the histologic lesions traditionally described as characteristic of BD in hearts procured from BD donors. METHODS: After BD diagnosis, specimens were taken from the left ventricle (n = 50) for histologic examination. Arterial blood samples were collected from 40 of the donors at different time-points (1 hour before BD; on BD diagnosis; and 1, 2, 3 and 4 hours after BD) to determine catecholamine levels by high-performance liquid chromatography (HPLC). RESULTS: The three hormones examined showed above-normal levels (epinephrine 2.36-fold, norepinephrine 8.56-fold, dopamine 54.76-fold). Release patterns included epinephrine and dopamine peaks at the time of BD and a norepinephrine peak 1 hour later. Fifty percent of the BD donors showed contraction bands and 62% displayed cardiomyocyte necrosis, which was associated with focal mononuclear cell infiltrates in 18% of cases. In 40% of donors, colocalized apoptotic and necrotic damage was observed. CONCLUSIONS: Differing extents of BD-associated cardiac lesions were observed in the donors, and >50% also showed apoptotic damage. The expected catecholamine peak at the time of BD was only detected for epinephrine and dopamine. Hormone increases were below those described in the literature, except for dopamine.

A molecular approach to apoptosis in the human heart during brain death.

BACKGROUND: Brain death induces changes in tissues and organs destined for transplant at the cell, molecular, and endocrine level including cell death through apoptosis. This study was designed to examine apoptotic damage in cardiac tissue obtained from brain dead donors. METHODS: Fifty tissue specimens from the left ventricles of individual donors were processed to evaluate changes in the expression levels of five genes involved in apoptosis (BAX, BCL2, CASPASE 3, CYTOCHROME C, and FAS) using the real time-polymerase chain reaction technique. Expression levels were quantified by the relative standard method and results normalized to the levels recorded for the endogenous control peptidylprolyl isomerase A. The HIF1alpha gene was also determined to check for the possibility of hypoxic damage. Control ventricular tissue specimens were obtained from patients undergoing mitral valve replacement. RESULTS: Using a mixed linear model it was determined that the sample type (donor vs. control patient) significantly affected (P<0.0001) expression levels of the genes examined reflected by their Ct values. Three of the genes (BAX, CASPASE 3, and FAS) showed significantly higher (Student's t test, P<0.05) expression levels (4.89-, 7.85-, and 12.14-fold endogenous control values, respectively) in donors compared with control patients (2.31-, 2.64-, and 3.57-fold endogenous control values, respectively) indicating the activation of apoptosis during brain death. CONCLUSION: Our findings suggest the possibility of using antiapoptosis agents to prevent cardiac injury and improve posttransplant behavior.