The involvement of oxidants and NF-κB in cytokine-induced MMP-9 synthesis by bone marrow-derived osteoprogenitor cells.

OBJECTIVE AND DESIGN: The activity of immune cells affects the balance between bone mineralization and resorption carried out by the opposing actions of osteoblasts and osteoclasts, respectively. This study was aimed at determining the possible interaction between inflammatory conditions and collagen type I degrading MMP (mainly MMP-2 and MMP-9) synthesis and secretion in rat osteoprogenitors. MATERIALS AND METHODS: The study was performed using primary rat bone marrow-derived osteoprogenitors during their advanced osteogenesis. Biochemical, immunohistochemical, and molecular biology techniques were used to investigate the influence of pro-inflammatory cytokines on MMP-2 and MMP-9 synthesis and secretion in osteoprogenitors. RESULTS: Results indicated that both synthesis and secretion of MMPs (MMP-1, -2, -8, -9, and -13) were significantly induced after pro-inflammatory cytokine treatments, except MMP-2, whose levels remained unchanged. NF-κB (nuclear factor kappa-light chain enhancer of activated B cells) inhibition assays showed that induced MMP-9 secretion by inflammatory cytokines was mediated by activation of NF-κB via the classical pathway and that oxidants play a significant role in this signal transduction pathway. In contrast, no such effect was observed for synthesis of MMP-2. CONCLUSIONS: These results indicate the possibility that inflammatory processes may trigger osteoblasts to absorb bone by secreting elevated levels of MMPs capable of degrading collagen type I, especially MMP-9 which is upregulated due to increased NF-κB transcription activity.

Priming integrin alpha5 promotes human mesenchymal stromal cell osteoblast differentiation and osteogenesis.

Adult human mesenchymal stromal cells (hMSCs) have the potential to differentiate into chondrogenic, adipogenic, or osteogenic lineages, providing a potential source for tissue regeneration. An important issue for efficient bone regeneration is to identify factors that can be targeted to promote the osteogenic potential of hMSCs. Using transcriptome analysis, we found that integrin alpha5 (ITGA5) expression is up-regulated during dexamethasone-induced osteoblast differentiation of hMSCs. Gain-of-function studies showed that ITGA5 promotes the expression of osteoblast phenotypic markers and in vitro osteogenesis of hMSCs. Down-regulation of endogenous ITGA5 using specific shRNAs blunted osteoblast marker gene expression and osteogenic differentiation. Molecular analyses showed that the enhanced osteoblast differentiation induced by ITGA5 was mediated by activation of focal adhesion kinase/ERK1/2-MAPKs and PI3K signaling pathways. Remarkably, activation of endogenous ITGA5 using agonists such as a specific antibody that primes the integrin or a peptide that specifically activates ITGA5 was sufficient to enhance ERK1/2-MAPKs and PI3K signaling and to promote osteoblast differentiation and osteogenic capacity of hMSCs. Importantly, we demonstrated that hMSCs engineered to overexpress ITGA5 exhibited a marked increase in their osteogenic potential in vivo. Taken together, these findings not only reveal that ITGA5 is required for osteoblast differentiation of adult hMSCs but also provide a targeted strategy using ITGA5 agonists to promote the osteogenic capacity of hMSCs. This may be used for tissue regeneration in bone disorders where the recruitment or capacity of hMSCs is compromised.

Analysis of progenitor cell-scaffold combinations by in vivo non-invasive photonic imaging.

Recent developments in stem cell research have promoted a flourishing of new biomaterials and scaffolds for tissue repair. However, there is a scarcity of procedures to monitor the performance of scaffold-stem cell combinations implanted in live animals, avoiding the inherent artefacts associated with in vitro assay conditions. We report the implementation of a procedure based on the use of the luciferase gene as a cell proliferation tracer to monitor, by in vivo non-invasive imaging, the performance of stem cell-biomaterial combinations used for tissue regeneration. In a model system using immunodepressed mice we show preference of a mouse embryonic mesenchymal cell line (C3H/10T1/2) for specific implantation sites and biomaterials during a prolonged in vivo growth period (3 months). Moreover, we analyzed the safety of implanted cells using a sensitive luminometric procedure and showed that the implanted cells did not spread to other organs. Our results demonstrate the utility of this simple and resource-saving procedure in the development and screening of biomaterials for tissue engineering.

Mandibular defect repair by TGF-beta and IGF-1 released from a biodegradable osteoconductive hydrogel.

PURPOSE: Bone regeneration is crucial in the healing of defects in the craniofacial complex. The ability of transforming growth factor-beta1 (TGF-beta1) and insulin-like growth factor-1 (IGF-1), incorporated into a hydrogel scaffold to induce bone regeneration, was evaluated in a rat mandible defect model. MATERIAL AND METHODS: Hydrogel scaffolds containing either transforming growth factor-beta1 (TGF-beta1), insulin-like growth factor-1 (IGF-1), TGF-beta+ IGF-1, or saline, were implanted in rat mandibular bone defects. In a control group the defects were treated by saline alone. Bone defect healing was tested after 3 and 6 weeks by radiology and morphology. RESULTS: Soft tissue radiographs indicated that the area of new bone formation increased gradually after 3 and at 6 weeks. The percentage of closure after 3 weeks was less than the percentage closure after 6 weeks. The amount of calcified material in the TGF-beta and TGF-beta+IGF-1-treated groups had increased more than in the saline-containing hydrogel and control (saline-treated) defects. The percentages of defect closures were 37, 38, 24, 14, and 11% after 3 weeks, and 94, 91, 84, 72, and 29% after 6 weeks, in the TGF-beta+IGF-1, TGF-beta, IGF-1, saline containing hydrogel and saline-treated animals, respectively. Three-dimensional computerized tomography (3D CT) images showed that the 3D shape of the bones was restored. Morphological analysis of the defects treated with hydrogel containing TGF-beta, IGF-1 or TGF-beta+IGF-1 revealed significant bone formation after 6 weeks. CONCLUSION: It is concluded that the hydrogel scaffold impregnated with growth factors can induce bone regeneration and is therefore a promising surgical tool for enhancement of surgical repair of bone defects.

CD10: a valuable tool for the light microscopic diagnosis of microvillous inclusion disease (familial microvillous atrophy).

Microvillous inclusion disease (MID) is a specific disorder of the intestinal brush border that leads to intractable secretory diarrhea in infants. At present, electron microscopic analysis is required for its definitive diagnosis. However, this technique is not always available or feasible, and the diagnostic microvillous inclusions may not be evident in all specimens. Accordingly, the availability of a panel of histochemical and immunohistochemical stains displaying a specific staining pattern for MID will allow pathologists to reach a definitive diagnosis of this disorder without recourse to electron microscopy. CD10 is a membrane-associated neutral peptidase, shown to have a linear brush-border staining pattern in normal small intestine. We studied the staining pattern of CD10 in small intestinal biopsies from six patients with MID and in 24 control cases (10 normal small intestine, 10 celiac disease, two autoimmune enteropathy, and two allergic enteropathy). All MID cases revealed prominent cytoplasmic CD10 immunoreactivity in surface enterocytes. In contrast, all control cases showed linear brush-border staining. Similar results were obtained with periodic acid-Schiff, polyclonal carcinoembryonic antigen, and alkaline phosphatase, three stains known to show cytoplasmic staining of surface enterocytes in MID. In conclusion, CD10 is a valuable tool for the diagnosis of MID. It may be used as part of a panel that includes other stains with a distinctive staining pattern in MID such as periodic acid-Schiff, polyclonal carcinoembryonic antigen, and alkaline phosphatase. We suggest that the definitive diagnosis of MID can be reached when small bowel biopsies from infants with intractable diarrhea display cytoplasmic staining of surface enterocytes with the above-mentioned stains.

Controlled release of BMP-2 from a sintered polymer scaffold enhances bone repair in a mouse calvarial defect model.

Sustained and controlled delivery of growth factors, such as bone morphogenetic protein 2 (BMP-2), from polymer scaffolds has excellent potential for enhancing bone regeneration. The present study investigated the use of novel sintered polymer scaffolds prepared using temperature-sensitive PLGA/PEG particles. Growth factors can be incorporated into these scaffolds by mixing the reconstituted growth factor with the particles prior to sintering. The ability of the PLGA/PEG scaffolds to deliver BMP-2 in a controlled and sustained manner was assessed and the osteogenic potential of these scaffolds was determined in a mouse calvarial defect model. BMP-2 was released from the scaffolds in vitro over 3 weeks. On average, ca. 70% of the BMP-2 loaded into the scaffolds was released by the end of this time period. The released BMP-2 was shown to be active and to induce osteogenesis when used in a cell culture assay. A substantial increase in new bone volume of 55% was observed in a mouse calvarial defect model for BMP-2-loaded PLGA/PEG scaffolds compared to empty defect controls. An increase in new bone volume of 31% was observed for PLGA/PEG scaffolds without BMP-2, compared to empty defect controls. These results demonstrate the potential of novel PLGA/PEG scaffolds for sustained BMP-2 delivery for bone-regeneration applications.

The generation of hybrid electrospun nanofiber layer with extracellular matrix derived from human pluripotent stem cells, for regenerative medicine applications.

Extracellular matrix (ECM) has been utilized as a biological scaffold for tissue engineering applications in a variety of body systems, due to its bioactivity and biocompatibility. In the current study we developed a modified protocol for the efficient and reproducible derivation of mesenchymal progenitor cells (MPCs) from human embryonic stem cells as well as human induced pluripotent stem cells (hiPSCs) originating from hair follicle keratinocytes (HFKTs). ECM was produced from these MPCs and characterized in comparison to adipose mesenchymal stem cell ECM, demonstrating robust ECM generation by the excised HFKT-iPSC-MPCs. Exploiting the advantages of electrospinning we generated two types of electrospun biodegradable nanofiber layers (NFLs), fabricated from polycaprolactone (PCL) and poly(lactic-co-glycolic acid) (PLGA), which provide mechanical support for cell seeding and ECM generation. Elucidating the optimized decellularization treatment we were able to generate an available "off-the-shelf" implantable product (NFL-ECM). Using rat subcutaneous transplantation model we demonstrate that this stem-cell-derived construct is biocompatible and biodegradable and holds great potential for tissue regeneration applications.

Low dose BMP-2 treatment for bone repair using a PEGylated fibrinogen hydrogel matrix.

Bone repair strategies utilizing resorbable biomaterial implants aim to stimulate endogenous cells in order to gradually replace the implant with functional repair tissue. These biomaterials should therefore be biodegradable, osteoconductive, osteoinductive, and maintain their integrity until the newly formed host tissue can contribute proper function. In recent years there has been impressive clinical outcomes for this strategy when using osteoconductive hydrogel biomaterials in combination with osteoinductive growth factors such as human recombinant bone morphogenic protein (hrBMP-2). However, the success of hrBMP-2 treatments is not without risks if the factor is delivered too rapidly and at very high doses because of a suboptimal biomaterial. Therefore, the aim of this study was to evaluate the use of a PEGylated fibrinogen (PF) provisional matrix as a delivery system for low-dose hrBMP-2 treatment in a critical size maxillofacial bone defect model. PF is a semi-synthetic hydrogel material that can regulate the release of physiological doses of hrBMP-2 based on its controllable physical properties and biodegradation. hrBMP-2 release from the PF material and hrBMP-2 bioactivity were validated using in vitro assays and a subcutaneous implantation model in rats. Critical size calvarial defects in mice were treated orthotopically with PF containing 8 µg/ml hrBMP-2 to demonstrate the capacity of these bioactive implants to induce enhanced bone formation in as little as 6 weeks. Control defects treated with PF alone or left empty resulted in far less bone formation when compared to the PF/hrBMP-2 treated defects. These results demonstrate the feasibility of using a semi-synthetic biomaterial containing small doses of osteoinductive hrBMP-2 as an effective treatment for maxillofacial bone defects.

Lentiviral-mediated integrin α5 expression in human adult mesenchymal stromal cells promotes bone repair in mouse cranial and long-bone defects.

Abstract Adult human mesenchymal stromal cells (hMSCs) are an important source for tissue repair in regenerative medicine. Notably, targeted gene therapy in hMSCs to promote osteogenic differentiation may help in the development of novel therapeutic approaches for bone repair. We recently showed that α5 integrin (ITGA5) promotes osteoblast differentiation in bone marrow-derived hMSCs. Here, we determined whether lentiviral (LV)-mediated expression of ITGA5 in hMSCs derived from the bone-marrow stroma of healthy individuals may promote bone repair in vivo in two relevant critical-size bone defects in the mouse. In a first series of experiments, control or LV-ITGA5-transduced hMSCs were seeded on collagen-based gelatin sponge and transplanted in a cranial critical-size defect (5 mm) in Nude-Foxn1nu mice. Microcomputed tomography and quantitative histological analyses after 8 weeks showed no or little de novo bone formation in defects implanted with collagen sponge alone or with hMSCs, respectively. In contrast, implantation of collagen sponge with LV-ITGA5-transduced hMSCs showed greater bone formation compared with control hMSCs. We also tested the bone-repair potential of LV-mediated ITGA5 expression in hMSCs in a critical-size long-bone defect (2 mm) in femur in Nude-Foxn1nu mice. Bone remnants were stabilized with external fixation, and control or LV-ITGA5-transduced hMSCs mixed with coral/hydroxyapatite particles were transplanted into the critical-size long-bone defect. Histological analysis after 8 weeks showed that LV-ITGA5-transduced hMSCs implanted with particles induced 85% bone regeneration and repair. These results demonstrate that repair of critical-size mouse cranial and long-bone defects can be induced using LV-mediated ITGA5 gene expression in hMSCs, which provides a novel gene therapy for bone regeneration.

Slow-release human recombinant bone morphogenetic protein-2 embedded within electrospun scaffolds for regeneration of bone defect: in vitro and in vivo evaluation.

The design of mat-like scaffolds slow-releasing bone morphogenetic protein-2 (BMP-2) retaining bone regeneration functions has been a major challenge in tissue engineering. This study aimed to develop core-shell fiber scaffolds releasing BMP-2 to support bone regeneration. BMP-2 was incorporated in an aqueous core solution of poly(ethylene oxide), whereas the shell solution was made of polycaprolactone blended with poly(ethylene glycol). This blending induced pores in the shell, which pronouncedly affected the movement of proteins out of the fibers. BMP-2 release profiles were monitored. In vitro bioactivity of BMP-2 released from the scaffolds was assessed using human mesenchymal stem cells by measuring alkaline phosphatase activity. Bone regeneration capabilities were demonstrated by implanting the BMP-2-embedded scaffolds in rat cranial defect model followed by micro-computed tomography analysis. The degree of fiber's shell porosity, highly correlative with the slow- and fast-release patterns of BMP-2, were found to be dependent on the relative amount of poly(ethylene glycol) within the shell. In vitro assays of scaffolds manifesting the slow-release pattern have revealed significant (∼9-fold) increase in alkaline phosphatase activity, compared to fast BMP-2 releasing scaffolds. Likewise, in vivo studies have revealed significant bone regeneration in cranial defects of scaffold implants with recombinant human BMP-2 with slow-release pattern.

A model for tissue engineering applications: femoral critical size defect in immunodeficient mice.

Animal models for preclinical functionality assays lie midway between in vitro systems such as cell culture and actual clinical trials. We have developed a novel external fixation device for femoral critical size defect (CSD) in the femurs of immunodeficient mice as an experimental model for studying bone regeneration and bone tissue engineering. The external fixation device comprises four pointed rods and dental acrylic paste. A segmental bone defect (2 mm) was created in the midshaft of the mouse femur. The CSD in the femur of the mice were either left untreated or treated with a bone allograft, a cell-scaffold construct, or a scaffold-only construct. The repair and healing processes of the CSD were monitored by digital x-ray radiography, microcomputed tomography, and histology. Repair of the femoral CSD was achieved with the bone allografts, and partial repair of the femoral CSD was achieved with the cell scaffold and the scaffold-only constructs. No repair of the nongrafted femoral CSD was observed. Our results establish the feasibility of this new mouse femoral model for CSD repair of segmental bone using a simple stabilized external fixation device. The model should prove especially useful for in vivo preclinical proof-of-concept studies that involve cell therapy-based technologies for bone tissue engineering applications in humans.

Cell-scaffold transplant of hydrogel seeded with rat bone marrow progenitors for bone regeneration.

Bone is the second most frequently transplanted tissue in humans and efforts are focused on developing cell-scaffold constructs which can be employed for autologous implantation in place of allogenic transplants. The objective of the present study was to examine the efficacy of a gelatin-based hydrogel scaffold to support osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells (MSCs) and its application in a cranial defect model. MSCs which were cultured on hydrogel under osteogenic conditions demonstrated typical osteogenic differentiation which included cluster formation with positive Alizarin Red S staining, sedimentation of calcium phosphate as defined by SEM and EDS spectroscopy and expression of mRNA osteogenic markers. Empty scaffolds or those containing either differentiated cells or naïve cells were implanted into cranial defects of athymic nude mice and the healing process was followed by µCT. Substantial bone formation (65%) was observed with osteogenic cell-scaffold constructs when compared to the naïve cell construct (25%) and the cell free scaffold (10%). Results demonstrated the potential of hydrogel scaffolds to serve as a supportive carrier for bone marrow-derived MSCs.

Human bone marrow mesenchymal stem cells: a systematic reappraisal via the genostem experience.

Genostem (acronym for "Adult mesenchymal stem cells engineering for connective tissue disorders. From the bench to the bed side") has been an European consortium of 30 teams working together on human bone marrow Mesenchymal Stem Cell (MSC) biological properties and repair capacity. Part of Genostem activity has been dedicated to the study of basic issues on undifferentiated MSCs properties and on signalling pathways leading to the differentiation into 3 of the connective tissue lineages, osteoblastic, chondrocytic and tenocytic. We have evidenced that native bone marrow MSCs and stromal cells, forming the niche of hematopoietic stem cells, were the same cellular entity located abluminally from marrow sinus endothelial cells. We have also shown that culture-amplified, clonogenic and highly-proliferative MSCs were bona fide stem cells, sharing with other stem cell types the major attributes of self-renewal and of multipotential priming to the lineages to which they can differentiate (osteoblasts, chondrocytes, adipocytes and vascular smooth muscle cells/pericytes). Extensive transcription profiling and in vitro and in vivo assays were applied to identify genes involved in differentiation. Thus we have described novel factors implicated in osteogenesis (FHL2, ITGA5, Fgf18), chondrogenesis (FOXO1A) and tenogenesis (Smad8). Another part of Genostem activity has been devoted to studies of the repair capacity of MSCs in animal models, a prerequisite for future clinical trials. We have developed novel scaffolds (chitosan, pharmacologically active microcarriers) useful for the repair of both bone and cartilage. Finally and most importantly, we have shown that locally implanted MSCs effectively repair bone, cartilage and tendon.

Engineering the growth factor microenvironment with fibronectin domains to promote wound and bone tissue healing.

Although growth factors naturally exert their morphogenetic influences within the context of the extracellular matrix microenvironment, the interactions among growth factors, their receptors, and other extracellular matrix components are typically ignored in clinical delivery of growth factors. We present an approach for engineering the cellular microenvironment to greatly accentuate the effects of vascular endothelial growth factor-A (VEGF-A) and platelet-derived growth factor-BB (PDGF-BB) for skin repair, and of bone morphogenetic protein-2 (BMP-2) and PDGF-BB for bone repair. A multifunctional recombinant fragment of fibronectin (FN) was engineered to comprise (i) a factor XIIIa substrate fibrin-binding sequence, (ii) the 9th to 10th type III FN repeat (FN III9-10) containing the major integrin-binding domain, and (iii) the 12th to 14th type III FN repeat (FN III12-14), which binds growth factors promiscuously, including VEGF-A165, PDGF-BB, and BMP-2. We show potent synergistic signaling and morphogenesis between α5ß1 integrin and the growth factor receptors, but only when FN III9-10 and FN III12-14 are proximally presented in the same polypeptide chain (FN III9-10/12-14). The multifunctional FN III9-10/12-14 greatly enhanced the regenerative effects of the growth factors in vivo in a diabetic mouse model of chronic wounds (primarily through an angiogenic mechanism) and in a rat model of critical-size bone defects (through a mesenchymal stem cell recruitment mechanism) at doses where the growth factors delivered within fibrin only had no significant effects.

Repair of full-thickness tendon injury using connective tissue progenitors efficiently derived from human embryonic stem cells and fetal tissues.

The use of stem cells for tissue engineering (TE) encourages scientists to design new platforms in the field of regenerative and reconstructive medicine. Human embryonic stem cells (hESC) have been proposed to be an important cell source for cell-based TE applications as well as an exciting tool for investigating the fundamentals of human development. Here, we describe the efficient derivation of connective tissue progenitors (CTPs) from hESC lines and fetal tissues. The CTPs were significantly expanded and induced to generate tendon tissues in vitro, with ultrastructural characteristics and biomechanical properties typical of mature tendons. We describe a simple method for engineering tendon grafts that can successfully repair injured Achilles tendons and restore the ankle joint extension movement in mice. We also show the CTP's ability to differentiate into bone, cartilage, and fat both in vitro and in vivo. This study offers evidence for the possibility of using stem cell-derived engineered grafts to replace missing tissues, and sets a basic platform for future cell-based TE applications in the fields of orthopedics and reconstructive surgery.

Isolation of progenitor cells from cord blood using adhesion matrices.

The aim of this study was to develop optimal conditions for selective adhesion and isolation of mesenchymal progenitor cells (MPCs) from cord blood and to determine their potential for osteogenic differentiation. Mononuclear cells (MNCs) were isolated by Ficoll-Paque gradient and plated onto 48-well culture plates precoated with: human or bovine collagen type I, human collagen type IV, fibronectin or matrigel. Cultures were incubated in alphaMEM containing fetal calf serum. Viability of the adherent cells was determined by alamarBlue(R) assay after 2, 3, and 4 weeks. After 4 weeks in culture, cells were typsinized and replated. Primary cultures were analyzed by histochemistry and third passage cells by FACS. Isolated fibroblast-like cells were cultured in the presence of osteogenic factors and differentiation determined by Alizarin Red S staining, RT-PCR and electron dispersive spectroscopy (EDS). MNCs adhered to all types of matrices with the greatest adhesion rates on fibronectin. These cells were CD45(+), CD105(+), CD14(+), CD49a(+), CD49f(+), CD44(+) and CD34(-). The highest incidence of progenitor cells (PC) was observed on fibronectin and polystyrene. Passages were CD45(-), CD14(-), CD34(-) and weakly CD105(+). Primary cultures expressed endothelial/macrophage RNA markers whether cultured on fibronectin or polystyrene and these markers decreased upon passage. The best osteogenic differentiation was observed in MPCs cultured in osteogenic medium containing vitamin D(3) and FGF9. These cells expressed the bone-related mRNA, collagen type I, core binding factor I (Cbfa I), osteocalcin and osteopontin. EDS of deposits produced by these cells demonstrated a calcium/phosphate ratio parallel to hydroxyapatite. It was concluded that fibronectin increased adhesion rates and isolation potential of cord blood mesenchymal progenitor cells.

3D scaffolds for bone marrow stem cell support in bone repair.

Bone tissue repair is one of the major concerns of regenerative medicine. The current need for tissue replacements has necessitated the development of a new science termed 'bone tissue engineering'. The basic organization of bone tissue requires the design and fabrication of a porous 3D structure or 'scaffold' to contain the bone-forming cells. This scaffold should be formulated from biocompatible, osteoconductive materials that are not immunoreactive. 3D scaffolds provide the necessary support for cells to proliferate and maintain their capacity to differentiate and scaffolds containing bone marrow-derived osteoprogenitors can be employed within implants to enhance bone repair. The complex construct is intended to mimic the native in vivo microenvironment and this demands construction of bioactive scaffolds that are also capable of supporting vascularization as well as cell proliferation and osteogenic differentiation. 3D bioactive scaffolds containing committed osteoprogenitors can provide a promising surgical tool for bone tissue engineering directed at orthopedic and cranio-maxillofacial clinical applications.

Isolation of progenitor cells from cord blood using adhesion matrices.

The aim of this study was to develop optimal conditions for selective adhesion and isolation of mesenchymal progenitor cells (PCs) from cord blood and to determine their potential for osteogenic differentiation. Mononuclear cells (MNCs) were isolated by Ficoll-Paque gradient and plated onto 48-well culture plates precoated with: human or bovine collagen type I, human collagen type IV, fibronectin or matrigel. Cultures were incubated in alphaMEM containing fetal calf serum. Viability of the adherent cells was determined by alamarBlue(R) assay after 2, 3, and 4 weeks. After 4 weeks in culture, cells were typsinized and replated. Primary cultures were analyzed by histochemistry and third passage cells by FACS. Isolated fibroblast-like cells were cultured in the presence of osteogenic factors and differentiation determined by Alizarin Red S staining, RT-PCR and electron dispersive spectroscopy (EDS). MNCs adhered to all types of matrices with the greatest adhesion rates on fibronectin. These cells were CD45(+), CD105(+), CD14(+), CD49a(+), CD49f(+), CD44(+) and CD34(-). The highest incidence of PCs was observed on fibronectin and polystyrene. Passages were CD45(-), CD14(-), CD34(-) and weakly CD105(+). Primary cultures expressed endothelial/macrophage RNA markers whether cultured on fibronectin or polystyrene and these markers decreased upon passage. The best osteogenic differentiation was observed in MPCs cultured in osteogenic medium containing Vit D(3) and FGF9. These cells expressed the bone-related mRNA, collagen type I, core binding factor I (Cbfa I), osteocalcin and osteopontin. EDS of deposits produced by these cells demonstrated a calcium/phosphate ratio parallel to hydroxyapatite. It was concluded that fibronectin increased adhesion rates and isolation potential of cord blood mesenchymal progenitor cells.

DT56a (Femarelle) stimulates bone formation in female rats.

OBJECTIVE: DT56a is a natural compound for the treatment of menopausal symptoms and osteoporosis. The aim of this study was to examine the effects of long term treatment (two months) with DT56a on the skeletal tissues of intact and ovariectomised (OVX) adult rats. DESIGN: Thirty rats were divided into two groups, in one of which the rats were ovariectomised. The rats in each group were then treated for two months with DT56a, oestrogen or vehicle. SETTING: University and hospital laboratories. POPULATION: Thirty rats. METHODS: Histomorphometric measurements of trabecular bone volume (expressed as a percentage of total bone volume), trabecular and cortical thickness and growth plate width were recorded by a computerised system. In addition, creatine kinase (CK)-specific activity, as marker of oestrogen receptor activation, was measured in skeletal tissues and in the uterus. MAIN OUTCOME MEASURES: The changes in the histomorphometric measurements. RESULTS: OVX rats developed noticeable signs of osteoporosis, namely, significant decrease in trabecular bone volume and in trabecular and cortical thickness. DT56a, like oestrogen, restored the bone structure measurements of all tested parameters in the OVX rats to the values obtained in the intact rats. In skeletal tissues, CK activity was elevated in both treatment groups. However, in the uterus DT56a did not activate oestrogen receptors while oestrogen did elevate CK activity. CONCLUSIONS: DT56a was as effective as oestrogen in reversing the bone changes caused by OVX in rats.

The effect of interleukin-8 on the viability of injected adipose tissue in nude mice.

Adipose tissue injection as a free graft for the correction of soft-tissue defects is a widespread procedure in plastic surgery. The main problem in achieving long-term soft-tissue augmentation is partial absorption of the injected fat and hence the need for overcorrection and re-injection. The purpose of this study was to improve the viability of the injected fat by the use of interleukin-8. The rationale for the use of interleukin-8 was its abilities to accelerate angiogenesis and attract inflammatory cells and fibroblasts, providing the injected adipocytes more feeding vessels and a well-established graft bed to enhance their viability. Human adipose tissue, obtained by suction-assisted lipectomy, was re-injected into the subcutis in the scalp of nude mice. Interleukin-8 (0.25 ng) was injected subcutaneously to the scalp as a preparation of the recipient site 24 hours before the fat injection and was added to the fat graft itself (25 ng per 1 cc of injected fat). In the control group, pure fat without interleukin-8 was injected and no interleukin-8 was added for the preparation of the recipient site. One cubic centimeter of fat was injected in each animal in both the study and control groups. There were 10 animals in each group. The animals were euthanized 15 weeks after the procedure. Graft weight and volume were measured and histologic evaluation was performed. In addition, triglyceride content and adipose cell sizes were measured as parameters for fat cells viability. Histologic analysis demonstrated significantly less cyst formation in the group treated with interleukin-8. No significant differences were found between the groups with regard to graft weight and volume or the other histologic parameters investigated. No significant differences were demonstrated in adipose cell sizes and their triglyceride content. In conclusion, less cyst formation, indicating improved quality of the injected fat, can be obtained by the addition of interleukin-8. Further studies of various dosages of interleukin-8 and their long-term effect are required before these encouraging results could be applied clinically.