Hydrolytic degradation and in vivo resorption of poly-l-lactic acid-chitosan biomedical devices in the parietal bones of Wistar rats.

OBJECTIVES: This study sought to describe events related to the degradation/resorption of a device composed of polylactic acid (PLA) after implantation into Wistar rats. METHODS: Five-millimeter-diameter PLA rigid scaffolds and flexible analogs were elaborated, bioactivated through culture with osteoblasts, and implanted into the parietal bones of adult Wistar rats after 15 days. After 3 months, the samples were recovered and analyzed via optical microscopy (histochemical techniques) and scanning electron microscopy. This research was approved by the animal ethics review committee of Universidad of Valle in Cali, Colombia, according to the endorsement of the ethics committee CEAS 001-016. RESULTS: Initially, there was surface erosion and fragmentation of the device, inducing an inflammatory response compatible with the foreign body reaction, in addition to the presence of a pseudocapsule and a mixed inflammatory infiltrate that was responsible for phagocytosis of the material. Regeneration of the defect via the apposition of new bone occurred simultaneously with resorption of the material. CONCLUSIONS: The results illustrated that the degradation/resorption of PLA occurs in a centripetal pattern.

Structural analysis of the frontal and parietal bones of the human skull.

Bone specimens were collected from the frontal and parietal bones of 4 adult, human skulls. The microstructure was characterized using microcomputed tomography (micro-CT) at about 6-µm resolution to map the change of porosity as a function of the depth, P(d), from the inner surface nearest to the brain to the outer surface nearest to the skin. A quantifiable method was developed using the measured P(d) to objectively distinguish between the three layers of the skull: the outer table, diploë , and inner table. The thickness and average porosity of each of the layers were then calculated from the measured porosity distributions, and a Gaussian function was fit to the P(d) curves. Morphological parameters were compared between the two bone types (frontal and parietal), while accounting for skull-to-skull variability. Parietal bones generally had a larger diploë accompanied by a thinner inner table. The arrangement of the porous vesicular structure within the outer table was also obtained with micro-CT scans with longer scan times, using enhanced parameters for higher resolution and lower noise in the images. From these scans, the porous structure of the bone appeared to be randomly arranged in the transverse plane, compared to the porous structure of the human femur, which is aligned in the loading direction.

The Morphogenesis of Cranial Sutures in Zebrafish.

Using morphological, histological, and TEM analyses of the cranium, we provide a detailed description of bone and suture growth in zebrafish. Based on expression patterns and localization, we identified osteoblasts at different degrees of maturation. Our data confirm that, unlike in humans, zebrafish cranial sutures maintain lifelong patency to sustain skull growth. The cranial vault develops in a coordinated manner resulting in a structure that protects the brain. The zebrafish cranial roof parallels that of higher vertebrates and contains five major bones: one pair of frontal bones, one pair of parietal bones, and the supraoccipital bone. Parietal and frontal bones are formed by intramembranous ossification within a layer of mesenchyme positioned between the dermal mesenchyme and meninges surrounding the brain. The supraoccipital bone has an endochondral origin. Cranial bones are separated by connective tissue with a distinctive architecture of osteogenic cells and collagen fibrils. Here we show RNA in situ hybridization for col1a1a, col2a1a, col10a1, bglap/osteocalcin, fgfr1a, fgfr1b, fgfr2, fgfr3, foxq1, twist2, twist3, runx2a, runx2b, sp7/osterix, and spp1/ osteopontin, indicating that the expression of genes involved in suture development in mammals is preserved in zebrafish. We also present methods for examining the cranium and its sutures, which permit the study of the mechanisms involved in suture patency as well as their pathological obliteration. The model we develop has implications for the study of human disorders, including craniosynostosis, which affects 1 in 2,500 live births.

Activation of FGF signaling mediates proliferative and osteogenic differences between neural crest derived frontal and mesoderm parietal derived bone.

BACKGROUND: As a culmination of efforts over the last years, our knowledge of the embryonic origins of the mammalian frontal and parietal cranial bones is unambiguous. Progenitor cells that subsequently give rise to frontal bone are of neural crest origin, while parietal bone progenitors arise from paraxial mesoderm. Given the unique qualities of neural crest cells and the clear delineation of the embryonic origins of the calvarial bones, we sought to determine whether mouse neural crest derived frontal bone differs in biology from mesoderm derived parietal bone. METHODS: BrdU incorporation, immunoblotting and osteogenic differentiation assays were performed to investigate the proliferative rate and osteogenic potential of embryonic and postnatal osteoblasts derived from mouse frontal and parietal bones. Co-culture experiments and treatment with conditioned medium harvested from both types of osteoblasts were performed to investigate potential interactions between the two different tissue origin osteoblasts. Immunoblotting techniques were used to investigate the endogenous level of FGF-2 and the activation of three major FGF signaling pathways. Knockdown of FGF Receptor 1 (FgfR1) was employed to inactivate the FGF signaling. RESULTS: Our results demonstrated that striking differences in cell proliferation and osteogenic differentiation between the frontal and parietal bone can be detected already at embryonic stages. The greater proliferation rate, as well as osteogenic capacity of frontal bone derived osteoblasts, were paralleled by an elevated level of FGF-2 protein synthesis. Moreover, an enhanced activation of FGF-signaling pathways was observed in frontal bone derived osteoblasts. Finally, the greater osteogenic potential of frontal derived osteoblasts was dramatically impaired by knocking down FgfR1. CONCLUSIONS: Osteoblasts from mouse neural crest derived frontal bone displayed a greater proliferative and osteogenic potential and endogenous enhanced activation of FGF signaling compared to osteoblasts from mesoderm derived parietal bone. FGF signaling plays a key role in determining biological differences between the two types of osteoblasts.

Differential effects of TGF-beta1 and TGF-beta3 on chondrogenesis in posterofrontal cranial suture-derived mesenchymal cells in vitro.

BACKGROUND: Transforming growth factor (TGF)-beta1 has been associated with cranial suture fusion, whereas TGF-beta3 has been associated with suture patency. The mouse posterofrontal suture, analogous to the human metopic suture, fuses through endochondral ossification. METHODS: TGF-beta1 and TGF-beta3 expression in the posterofrontal suture was examined by immunohistochemistry. Next, the authors established cultures of suture-derived mesenchymal cells from the posterofrontal suture and examined the cellular responses to TGF-beta1 and TGF-beta3. Proliferation in response to TGF-beta isoforms was examined by bromodeoxyuridine incorporation. High-density micromass culture of posterofrontal mesenchymal cells was used to study the effect of TGF-beta1 and TGF-beta3 on chondrogenic differentiation. RESULTS: TGF-beta1 but not TGF-beta3 protein was highly expressed in chondrocytes within the posterofrontal suture. Significant increases in posterofrontal cell proliferation were observed with TGF-beta3 but not TGF-beta1. TGF-beta1 led to significant increases in chondrogenic-specific gene expression (including Sox9, Col II, Aggrecan, and Col X) as compared with moderate effects of TGF-beta3. TGF-beta1 increased cellular adhesion molecule expression (N-cadherin and fibronectin) and promoted cellular condensation, whereas TGF-beta3 increased cellular proliferation (PCNA expression). Finally, TGF-beta1 and, to a lesser extent, TGF-beta3 induced the expression of fibroblast growth factors (FGF-2 and FGF-18). CONCLUSIONS: TGF-beta1 and TGF-beta3 exhibit marked differences in their effects on chondrogenesis in posterfrontal suture-derived mesenchymal cells, influencing different stages of chondrogenic differentiation. TGF-beta3 significantly increased cellular proliferation, whereas TGF-beta1 induced precartilage condensation, promoting chondrocyte differentiation.

Molecular and cellular characterization of mouse calvarial osteoblasts derived from neural crest and paraxial mesoderm.

BACKGROUND: Cranial skeletogenic mesenchyme is derived from two distinct embryonic sources: mesoderm and cranial neural crest. Previous studies have focused on molecular and cellular differences of juvenile and adult osteoblasts. METHODS: To further understand the features of mouse-derived juvenile osteoblasts, the authors separated calvarial osteoblasts by their developmental origins: frontal bone-derived osteoblasts from cranial neural crest, and parietal bone-derived osteoblasts from paraxial mesoderm. Cells were harvested from a total of 120 mice. RESULTS: Interestingly, the authors observed distinct morphologies and proliferation potential of the two populations of osteoblasts. Osteogenic genes such as alkaline phosphatase, osteopontin, collagen I, and Wnt5a, which was recently identified as playing a role in skeletogenesis, were abundantly expressed in parietal bone-derived osteoblasts versus frontal bone-derived osteoblasts. In addition, fibroblast growth factor (FGF) receptor 2, and FGF-18 were more highly expressed in the parietal bone-derived osteoblasts, suggesting a more differentiated phenotype. In contrast, FGF-2, and adhesion molecules osteoblast cadherins and bone morphogenetic protein receptor IB, the bone tissue-specific type receptor were overexpressed in frontal bone-derived osteoblasts compared with parietal bone-derived osteoblasts. CONCLUSIONS: The authors observed that although neural crest-derived osteoblasts represented a population of less differentiated, faster growing cells, they formed bone nodules more rapidly than parietal bone-derived osteoblasts. This in vitro study suggests that embryonic tissue derivations influence postnatal in vitro calvarial osteoblast cell biology.

Reparative process in bone tissue of old animals.

Plastic repair of the trephination hole in the parietal bone with syngeneic cartilage was carried out in old rabbits. The ossification process eventuated in the formation of a callus by day 28 postoperation, while in control animals bone tissue defect still persisted during this period.

Transcriptional expression of calvarial bone after treatment with low-intensity ultrasound: an in vitro study.

The capacity to reossify a calvarial bone defect is very small in mature animals and in humans greater than 2 y of age. The clinical treatment of injured tissue sites of bones by low-intensity pulsed ultrasound is widespread, but little is known about the precise effects of ultrasound on the fundamental processes that promote repair and regeneration. In this study, we used real-time polymerase chain reaction (RT-PCR) to investigate the expression of osteogenesis-associated genes after stimulation by low-intensity ultrasound in adult mouse osteoblast from the parietal calvaria. The gene associated with the Runx2 pathway had notably higher levels after 1, 2 and 3 days of stimulation. Therefore, low-intensity ultrasound seems to have an effect on the transcriptional gene expression of the calvarial bone in vitro.

FGF9 can induce endochondral ossification in cranial mesenchyme.

BACKGROUND: The flat bones of the skull (i.e., the frontal and parietal bones) normally form through intramembranous ossification. At these sites cranial mesenchymal cells directly differentiate into osteoblasts without the formation of a cartilage intermediate. This type of ossification is distinct from endochondral ossification, a process that involves initial formation of cartilage and later replacement by bone. RESULTS: We have analyzed a line of transgenic mice that expresses FGF9, a member of the fibroblast growth factor family (FGF), in cranial mesenchymal cells. The parietal bones in these mice show a switch from intramembranous to endochondral ossification. Cranial cartilage precursors are induced to proliferate, then hypertrophy and are later replaced by bone. These changes are accompanied by upregulation of Sox9, Ihh, Col2a1, Col10a1 and downregulation of CbfaI and Osteocalcin. Fate mapping studies show that the cranial mesenchymal cells in the parietal region that show a switch in cell fate are likely to be derived from the mesoderm. CONCLUSION: These results demonstrate that FGF9 expression is sufficient to convert the differentiation program of (at least a subset of) mesoderm-derived cranial mesenchyme cells from intramembranous to endochondral ossification.

[The effect of serum rich in platelet-released growth factors on proliferation of rat osteoblast in vitro].

OBJECTIVE: To study the effect of serum rich in platlelet-released growth factors on the biological functions of rat osteoblast. METHODS: Rat osteoblasts were isolated from parietal bone of fetal Spraque-Dawley (SD) rat. Platelet-rich plasma(PRP) and platelet-poor plasma (PPP) obtained from healthy SD rat were activated by thrombin to get serum rich in growth factors (SRGF) or serum poor in growth factors (SPGF). The cells were treated with 1.25%, 2.5%, 5% SRGF or SPGF, and cellular mitogenic activity was evaluated by thiazoly blue (MTT) colorimetric assay. Results When compared with SPGF at the same concentration, SRGF promoted the proliferation of rat osteoblast significantly (P = 0.002 - 0.004), and the proliferation seemed to correlate with the concentration of SRGF (Spearman's correlation coefficient, r(s) = 0.834). CONCLUSION: SRGF is capable of upregulating the proliferation of rat osteoblast.

Effect of naringin collagen graft on bone formation.

Naringin is a flavonoid available commonly in citrus fruits and is also a HMG-CoA reductase inhibitor. Our laboratory compared the amount of new bone produced by naringin in collagen matrix to that produced by bone grafts and collagen matrix. Twenty bone defects, 5 mm x 10 mm were created in the parietal bone of 14 New Zealand White rabbits. In the experimental group, 5 defects were grafted with naringin solution mixed with collagen matrix, 5 defects were grafted with autogenous endochondral bone. In the control groups, 5 defects were grafted with collagen matrix alone (active control) and 5 were left empty (passive control). Animals were killed on day 14 and the defects were dissected and prepared for histological assessment. Serial sections were cut across each defect. Quantitative analysis of new bone formation was made on 150 sections (50 sections for each group) using image analysis. A total of 284% and 490% more new bone was present in defects grafted with naringin in collagen matrix than those grafted with bone and collagen, respectively. No bone was formed in the passive control group. In conclusion, naringin in collagen matrix have the effect of increasing new bone formation locally and can be used as a bone graft material.

Comparison of osteoblast spreading on microstructured dental implant surfaces and cell behaviour in an explant model of osseointegration. A scanning electron microscopic study.

OBJECTIVES: To compare interactions between rat calvarial osteoblasts and titanium dental implants with different microstructured surfaces. MATERIAL AND METHODS: Seven commercially available implants were used. Surfaces included plasma-sprayed, grit-blasted and/or acid-etched, smooth-machined and anodised titanium. Two methods were used to compare cell behaviour: (1) A cell-spreading assay in which percentages of cells at four different stages of attachment were identified by scanning electron microscopy and quantified within a 30 min attachment period. (2) Implants were placed in 'pocket culture' within nylon mesh sacs in contact with explanted calvarial bone fragments for 2 and 4 weeks. RESULTS: Surfaces combining grit blasting and acid etching, of microporous topography, showed significantly enhanced rates of cell spreading in comparison with the others. Differential cell morphology was observed in both suspension assays and pocket cultures. In the latter, cells migrated onto all surfaces. Multicellular layers with extracellular matrix (ECM) were present between the layers and on the material surfaces after 2 weeks. After 4 weeks, cell layers were more consolidated, and microstructures were obscured by layers of cells and ECM. Mineralised tissue was seen in association with ECM on grit-blasted surfaces of rough and smooth microtopography. CONCLUSIONS: The two methods provided complementary information: a rough surface of porous microstructure may enhance the rate of cell spreading. Differentiation and calcification occurred on surfaces of both rough and smooth microstructure.

Simvastatin, an HMG-CoA reductase inhibitor, reduced the expression of matrix metalloproteinase-9 (Gelatinase B) in osteoblastic cells and HT1080 fibrosarcoma cells.

MMP-9 or Gelatinase B, a member of the matrix metalloproteinase family (MMPs), plays important roles in physiological events such as tissue remodeling and in pathological processes that lead to destructive bone diseases, including osteoarthritis and periodontitis. In addition to its effect on the increase of total bone mass, statin (an HMG-CoA reductase inhibitor) suppresses the expression of MMPs. In this study, we proposed that simvastatin reduces MMP-9 expression in osteoblasts and HT1080 fibrosarcoma cell line. Gelatin zymography, Western blot analysis and reverse transcriptase-PCR were used to investigate the effects of simvastatin on MMP-9 in primary calvaria cells, U2-OS osteosarcoma cells, and HT1080 fibrosarcoma cells. The results from gelatin zymography and Western blot analysis revealed that simvastatin suppressed MMP-9 activity in these cells in concentration- and time-dependent manners. The effective concentrations of simvastatin were 100 - 500 nM, 5 - 15 microM, and 2.5 - 10 microM in primary calvaria, U2-OS, and HT1080 cells, respectively. Collectively, these results suggest that simvastatin is a potent drug for inhibition of MMP-9 expression in osteoblastic cells and HT1080 fibrosarcoma cells.

Synthetic extracellular matrices for in situ tissue engineering.

Cell interactions with the extracellular matrix play important roles in guiding tissue morphogenesis. The matrix stimulates cells to influence such things as differentiation and the cells actively remodel the matrix via local proteolytic activity. We have designed synthetic hydrogel networks that participate in this interplay: They signal cells via bound adhesion and growth factors, and they also respond to the remodeling influence of cell-associated proteases. Poly(ethylene glycol)-bis-vinylsulfone was crosslinked by a Michael-type addition reaction with a peptide containing three cysteine residues, the peptide sequence being cleavable between each cysteine residue by the cell-associated protease plasmin. Cells were able to invade gel networks that contained adhesion peptides and were crosslinked by plasmin-sensitive peptides, while materials lacking either of these two characteristics resisted cell infiltration. Incorporated bone morphogenetic protein-2 (BMP-2) induced bone healing in a rat model in materials that were both adhesive and plasmin-sensitive, while materials lacking plasmin sensitivity resisted formation of bone within the material. Furthermore, when a heparin bridge was incorporated as a BMP-2 affinity site, mimicking yet another characteristic of the extracellular matrix, statistically improved bone regeneration was observed.

Tissue response to a newly developed calcium phosphate cement containing succinic acid and carboxymethyl-chitin.

We developed a new calcium phosphate cement containing succinic acid and carboxymethyl-chitin in the liquid component. In this study, the biocompatibility and osteoconductivity of this new cement were investigated. After mixing, cement in putty form was implanted immediately between the periosteum and parietal bone and in the subcutaneous tissues of rats. In control cement, distilled water was used instead of the liquid component. In addition to histological evaluations, analyses with X-ray diffraction and Fourier transform infrared were performed for the subcutaneously implanted cements. Histological examination showed slight inflammation around the new cement on the bone and in the subcutaneous tissue at 1 week after surgery. At 2 weeks, the cement was partially bound to the parietal bone. The extent of the surface of the new cement directly in contact with the bone increased with time, and most of the undersurface of the new cement bound to the host parietal bone by 8 weeks. Analysis by X-ray diffraction showed that the new cement in the subcutaneous tissue was transformed into hydroxyapatite by 8 weeks. These results indicate that this new calcium phosphate cement is useful as a bone substitute material.

Effect of richlocaine on proliferative activity of osteoblasts and intracellular calcium content in rats.

We studied the effect of local anesthetic richlocaine on proliferation and intracellular calcium content in cultured osteoblasts from rat parietal bone. In a concentration of 1 mg/ml this drug produced a cytotoxic effect on osteoblasts. In concentrations of 0.01 and 0.001 mg/ml richlocaine in the absence and presence of subtoxic dose of sodium cyanide (0.2 mM) increased the number of osteoblasts by 15.4 and 36.6 or 13.8 and 38.6%, respectively. In a concentration of 1 mg/ml, richlocaine increased the content of cytosolic calcium in osteoblasts by 105%. These effects of richlocaine in low concentrations (0.01 and 0.001 mg/ml) can be related to stimulation of metabolic processes in osteoblasts.

Postparietal and prehatching ontogeny of the supraoccipital in Alligator mississippiensis (Archosauria, Crocodylia).

The first record of the postparietal bone of Alligator mississippiensis, documented by transverse histological sections, is presented. It is the first evidence of the presence of this bone within Recent reptiles. The postparietal is present in a specimen with a head length of 32.3 mm. The bone is a small dermal plate lying ventrally and posteriorly to the posterior margin of the parietal and dorsally to the trabecular bone, forming a dorsal surface of the supraoccipital portion of the neural endocranium. The trabecular bone develops perichondrally from the dorsal surface of the tectal cartilaginous bridge spanning between the dorsal portions of the otic capsules and occipital pilae. The bridge probably represents the fused tectum synoticum posterior plus tectum posterius. Later in ontogeny, the bridge ossifies endochondrally. The endochondrally ossifying bridge together with its perichondrally ossifying trabecular bone form the future supraoccipital. The trabecular bone is the integral part of the cranial endoskeleton and ontogenetically distinct from the dermal postparietal bone.

Osteoprotegerin is produced when prostaglandin synthesis is inhibited causing osteoclasts to detach from the surface of mouse parietal bone and attach to the endocranial membrane.

Osteoclast differentiation and activation is controlled, at least in part, by the counterbalancing influences of osteoprotegerin ligand (OPGL) and osteoprotegerin (OPG). Nonsteroidal anti-inflammatory drugs have been shown to inhibit bone loss in vivo and bone resorption in vitro, and this is associated with a loss of osteoclasts from the bone surface. We test the hypothesis that OPG mediates the inhibition of osteoclast activity that occurs with indomethacin in the mouse calvaria. Recombinant human OPG, like indomethacin, was found to cause osteoclasts to detach from the bone surface and attach to the adjacent endocranial membrane (periosteum). Recombinant human OPG also inhibited the stimulatory effect of prostaglandin E2 (PGE2), parathyroid hormone (PTH), and 1,25-dihydroxyvitamin D3 (1,25D3) on osteoclast adhesion to bone after an incubation with indomethacin. A function-blocking antibody to OPG and soluble human OPGL both inhibited the effect of indomethacin, leaving active osteoclasts on the bone. OPG activity was detected in the culture medium from indomethacin-treated bones and PTH, PGE2, 1,25D3, and dexamethasone all inhibited the production of OPG activity. We conclude that, in the absence of specific stimulators of bone resorption, OPG is produced by the mouse calvaria in vitro, which inhibits bone resorption by causing osteoclasts to detach from the bone surface.

Surfaces designed to control the projected area and shape of individual cells.

Materials with spatially resolved surface chemistry were designed to isolate individual mammalian cells to determine the influence of projected area on specific cell functions (e.g., proliferation, cytoskeletal organization). Surfaces were fabricated using a photolithographic process resulting in islands of cell binding N-(2-aminoethyl)-3-aminopropyl-trimethoxysilane (EDS) separated by a nonadhesive interpenetrating polymer network [poly (acrylamide-co-ethylene glycol); P (AAm-co-EG)]. The surfaces contained over 3800 adhesive islands/cm2, allowing for isolation of single cells with projected areas ranging from 100 microns 2 to 10,000 microns 2. These surfaces provide a useful tool for researching how cell morphology and mechanical forces affect cell function.