[Bone regeneration, cell therapy and tissue engineering].
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Animals↗
Biomedical subjects
J A Santamaría
Publications and source records attributed to J A Santamaría.
Production of a recombinant human basic fibroblast growth factor with a collagen binding domain.
Basic fibroblast growth factor (bFGF) is a potent in vitro mitogen for capillary endothelial cells, stimulates angiogenesis in vivo, and may participate in tissue repair. Basic FGF is found in abundance in tissues such as brain, kidney, and cartilage. This study reports the expression, purification, and renaturation of a biologically active human basic fibroblast growth factor fusion protein (hbFGF-F1) from Escherichia coli. A prokaryotic expression vector was engineered to produce a tripartite fusion protein consisting of a purification tag, a protease-sensitive linker and collagen binding domain, and a cDNA sequence encoding the active fragment of hbFGF. The expressed hbFGF-F1 and hbFGF-F2 (it contains the collagen binding domain), located in inclusion bodies, were solubilized with 6 M guanidine-HCl and renatured by a glutathione redox system and protracted dialysis under various experimental conditions. The purification of the recombinant proteins was achieved by binding the His-tag of the fusion protein on a nickel-nitrilotriacetic acid metal chelate column. The biological activity of the recombinant growth factor was demonstrated by its ability to stimulate proliferation of human vein endothelial cells, monitored by [3H]thymidine incorporation, where commercial recombinant human bFGF (rhbFGF) served as a positive control. Purified rhbFGF-F1 and rhbFGF-F2 constructs exhibited proliferative activity comparable to commercial rhbFGF. The high-affinity binding was demonstrated by the binding of [3H]collagen to the rhbFGF-F2 protein immobilized on a Ni-nitrilotriacetic acid column. The rhbFGF-F2 fusion protein bound to collagen-coated surfaces with high affinity. Taken together, these results demonstrate that biologically active rhbFGF fusion proteins can be recovered from transformed bacteria by oxidative refolding; thus, providing a means for their high-yield production, purification, and renaturation from microorganisms. Furthermore, we demonstrate that the auxiliary collagen binding domain effectively targets the recombinant growth factor to type I collagen. These studies advance the technology necessary to generate large quantities of targeted bFGF fusion proteins for specific biomedical applications.
Collagen↗
Selection and amplification of a bone marrow cell population and its induction to the chondro-osteogenic lineage by rhOP-1: an in vitro and in vivo study.
The differentiation and maturation of osteoprogenitor cells into osteoblasts are processes which are thought to be modulated by transforming growth factors-beta (TGF-beta) as well as by bone morphogenetic proteins (BMPs). Osteogenic protein-1 (OP-1, also known as BMP-7) is a member of the BMP family, and it is considered to have important regulatory roles in skeletal embryogenesis and bone healing. Rat bone marrow cells were cultured in vitro in a collagen-gel medium containing 0.5% fetal bovine serum (FBS) for 10 days in the presence of 40 ng/ml recombinant human OP-1 (rhOP-1). Under these conditions, survival of the bone marrow cell population was dependent on the presence of rhOP-1. Subsequently, the selected cells were cultured-for 6 days in medium containing 40 ng rhOP-1 and 10% FBS. During the last 2 days, dexamethasone (10(-8) M) and beta-glycerophosphate (2 mM) were added to potentiate osteoinduction. Concomitant with an up-regulation of cell proliferation, DNA synthesis levels, colony number and size were determined. Chondro-osteogenic differentiation in vitro was evaluated in terms of the expression of alkaline phosphatase, the production of osteocalcin and the formation of mineralized matrix. After culturing in vitro, cells were placed inside diffusion chambers or inactivated demineralized bone matrix (DBM) cylinders and implanted subdermically into the backs of old rats for 28 days. Biochemical, histological and immunocytochemical analyses provided evidence of cartilage and osteoid tissue inside the diffusion chambers, whereas bone was also observed inside the DBM implants. In conclusion, this experimental procedure is capable of selecting a cell population from bone marrow which, in the presence of rhOP-1, achieves skeletogenic potential under in vitro as well as in vivo environments.
Alkaline Phosphatase↗
Eikenella corrodens skull infection: a case report with review of the literature.
BACKGROUND: Subgaleal abscesses and skull osteomyelitis are very uncommon since the introduction of antibiotics. Eikenella corrodens infection is extremely rare in childhood and has never been reported in calvarial osteomyelitis. METHODS: We present a previously healthy 9-year-old boy, with a history of frontal contusion without injury, who developed E corrodens osteomyelitis of the skull. The radiographic findings are reviewed, including skull films and computed tomographic scans. The patient underwent surgical debridement of the lesion, as well as systemic medical therapy with amoxicillin. We review the medical and surgical therapy for such lesions. The differential diagnosis of a posttraumatic scalp swelling is also reviewed. RESULTS: Good resolution after debridement and antibiotic therapy is reported. CONCLUSIONS: Surgical intervention is emphasized for the removal of bony sequestrum and nonviable bone while maintaining an intact dura.
Child↗
Incorporation of bromodeoxyuridine in regenerating fin tissue of the goldfish Carassius auratus.
We have investigated the pattern of incorporation of 5-bromo-2'-deoxyuridine-5'-monophosphate (BrdU) by proliferating cells during regeneration of the tail fin of Carassius auratus. Fifteen days after amputation, intraperitoneal injection of a single dose of 0.25 mg/g wet weight of BrdU and subsequent immunocytochemical detection on sections revealed groups of replicating cells in the blastema and epidermis at different proximodistal levels. Proliferating blastemal cells were confined to a crowded, compact distal area that lost its replicative capacity laterally, causing the differentiation of scleroblasts, which synthesize the lepidotrichia hemisegments. Proximally, but centrally located, the blastemal cells did not incorporate BrdU and they differentiated giving rise to the mature intraray connective tissue. An independent cell-proliferation process was noted in the epidermis. The distal cap did not proliferate; the lateral faces of the epidermis showed high rates of cell replication in the central layer at every level of the regenerate rays; quiescent cells remained in the superficial layers. The basal epidermal cells did not incorporate BrdU when actinotrichia were present. The possible role of basal epidermal cells in the synthesis of actinotrichia, the contribution of these collagen macrofibrils to the morphogenetic process, and the different pathways of cell differentiation during fin regeneration are discussed.
Animals↗
Cell proliferation in fin fish regeneration.
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Animals↗
Perinotochordal connective sheet of gilthead sea bream larvae (Sparus aurata, L.) affected by axial malformations: an histochemical and immunocytochemical study.
BACKGROUND: Spinal malformations in adult teleosts occur under natural conditions and, more frequently, in culture exploitations. Skeletal deformities are linked with dysfunctions in collagen metabolism. We studied axial deviations appearing in early larval stages of cultured sea bream (Sparus aurata, L.). METHODS: To evaluate connective tissue components of normal and lordotic fish we used histochemistry (alcian blue, picrosirius-polarization, clorhydric orcein, fuchsin resorcin), immunohistochemistry (anti-collagen I, II, III, and IV), and specific enzymatic digestions. The results were evaluated by semiquantitative methods. RESULTS: Lordosis appeared before a vertebral column was developed, thus affecting the only skeletal structure present in the animal body, the notochord. At this stage the animal depends on the vitelline sac and an inflated swim-bladder is missing. The region of the curvature showed strong alterations in the arrangement of the muscle bundles and irregularities in notochord and perinotochordal collagen sheet. Histochemical and immunocytochemical analysis of the perinotochordal sheet revealed the presence of type II collagen, non-sulfated glycosaminoglycans, and elastic fibers in normal and lordotic specimens. Low collagen-proteoglycan interactions occurred in lordotic animals. CONCLUSIONS: Lordosis in Sparus aurata originated during embryonic development and was characterized by disorganized connective tissue and muscle bundles. No major differences in connective tissue constituents were seen with respect to normal specimens.
Animals↗
Interactions of the lepidotrichial matrix components during tail fin regeneration in teleosts.
Teleost fin rays are able to regenerate, when they are cut, restoring the whole structure in a few weeks. Following the formation and growth of an apical blastema, deposition of lepidotrichial matrix occurs. We have histo and immunochemically analyzed the maturation process of the lepidotrichial hemisegment, pointing out the interactions between their components and likewise the temporal and spatial distribution of some extracellular matrix components during regeneration. Lepidotrichial matrix is rich in sulfated glycosaminoglycans (GAGs), most of which are forming proteoglycans. Collagen is abundant and it strongly interacts with GAGs, as the tissue differentiates. The use of specific digestions with papain and collagenase suggests that some mannose rich glycoproteins may be also implicated in lepidotrichial maturation before mineralization. In each hemisegment a central band (CB) can be observed. In spite of the histochemical similarities between the CB and the subepidermical basement membrane, neither collagen IV nor laminin are present. This CB could be the result of a transient transdifferentiation of the outer lepidotrichial synthesizing cells.
Animals↗
Tail fin regeneration in teleosts: cell-extracellular matrix interaction in blastemal differentiation.
The fins of teleosts are appendices capable of regenerating by an epimorphic process that completely restores in a few days the original shape and size. After a partial amputation, the process that follows consists of: wound healing, blastema formation and cell differentiation to form a particular cell population responsible for the building of the different tissue elements. The present work deals with the histochemical and ultrastructural characterisation of the extracellular matrix (ECM) components in different blastemal regions. In the subepidermal space of the tip of a regenerating ray, two zones can be identified: the blastema proper (BP) that occupies the more apical region and extends proximally along the centre of the regenerating ray, and the actinotrichial blastemic region (ABR), located laterally to the former, about some micrometres beneath the tip and tightly related with the palisade of the actinotrichia. Hyaluronate is the more prominent glycosaminoglycan (GAG) in BP whereas sulphated GAGs and collagen are scarce in distal portions but they can be identified proximally. In the ABR a network composed mainly of sulphated GAGs and in part of collagen is developed. This network connects the actinotrichia of each palisade and extends around them. The significance of the ECM components in the morphogenetic processes of the regenerating rays is discussed. In addition a model for the synthesis of lepidotrichia by lepidotrichium-forming cells, differentiated from blastemal cells, is given.
Animals↗