Prostaglandins differentially affect osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells.

Adipose tissue-derived mesenchymal stem cells (AT-MSCs) are currently used for bone tissue engineering. AT-MSCs undergoing osteogenic differentiation respond to mechanical loading with increased cyclooxygenase-2 gene expression, a key enzyme in prostaglandin (PG) synthesis. PGs are potent multifunctional regulators in bone, exhibiting stimulatory and inhibitory effects on bone formation and resorption. PGE(2), but not PGI(2) or PGF(2), recruits osteoprogenitors from the bone marrow space and influences their differentiation. We hypothesize that PGE(2), PGI(2), and PGF(2) may differentially regulate osteogenic differentiation of human AT-MSCs. PGE(2), PGI(2), and PGF(2) (0.01-10 microM) affected osteogenic differentiation, but not proliferation of AT-MSCs after 4-14 days. Only PGF(2) (0.01-10 microM) increased alkaline phosphatase (ALP) activity at day 4. PGE(2) (10 microM), PGI(2) (0.01-10 microM), and PGF(2) (10 microM) decreased ALP activity, whereas PGF(2) (0.1 microM) increased ALP activity at day 14. PGF(2) (0.01-0.1 microM) and PGI(2) (0.01 microM) upregulated osteopontin gene expression, and PGF(2) (0.01 microM) upregulated alpha1(I)procollagen gene expression at day 4. PGE(2) and PGF(2) (10 microM) at day 4 and PGF(2) (1 microM) at day 14 downregulated runt-related transcription factor-2 gene expression. We conclude that PGE(2), PGI(2), and PGF(2) differentially affect osteogenic differentiation of AT-MSCs, with PGF(2) being the most potent. Thus, locally produced PGF(2) might be most beneficial in promoting osteogenic differentiation of AT-MSCs, resulting in enhanced bone formation for bone tissue engineering.

Salivary agglutinin/DMBT1SAG expression is up-regulated in the presence of salivary gland tumors.

Salivary agglutinin (SAG) is encoded by the gene Deleted in Malignant Brain Tumors 1 (DMBT1) and represents the salivary variant of DMBT1 (DMBT1(SAG)). While SAG is a bona fide anti-caries factor, DMBT1 was proposed as a candidate tumor-suppressor for brain, digestive tract, and lung cancer. Though DMBT1(SAG) is expressed in the salivary glands, its expression in salivary gland tumors is unknown. Here we analyzed DMBT1(SAG) expression in 20 salivary gland tumors and 14 tumor-flanking tissues by immunohistochemistry. DMBT1(SAG) in salivary gland tumors resembles the changes of expression levels known from DMBT1 in tumors in other cancer types. Particularly, DMBT1(SAG) was up-regulated in 10/14 tumor-flanking tissues, and a strong staining of the luminal content in the tumor and/or the tumor-flanking tissue was observed in 14/20 cases. This suggests that, in addition to its role in caries defense, SAG may serve as a potential tumor indicator and/or tumor suppressor in salivary gland tissue.

Development of transplanted pulp tissue containing epithelial sheath into a tooth-like structure.

The aim of these studies was to find out whether intact neonatal pulp tissue containing residual epithelial cells can induce the development of a tooth-like structure in situ. First maxillary neonatal hamster molar pulps containing adhering undifferentiated epithelial cells were transplanted submucosally in the oral cavity of recipient mothers for periods ranging from 2-8 weeks and the tissues were then processed for light microscopy. Developing tooth-like structures containing mineralised tubular dentine, predentine and a vascularised pulp-like chamber lined with functional odontoblast-like cells were observed in the specimens within 2 weeks of transplantation. Enamel and root formation were not observed. These data indicate that neonatal dental pulp tissues containing epithelial cell remnants have the capacity to develop into tooth-like structures and that this could be the explanation for the development of tooth-like structures sometimes observed in infants after extraction of a natal tooth.

Bone tissue response to biodegradable polymers used for intra medullary fracture fixation: a long-term in vivo study in sheep femora.

Since the degradability of the synthetic polymers used for fracture fixation is still unclear, a research project with biodegradable interlocking nails with a longterm implantation period has been started. In 21 female sheep a complete mid-shaft osteotomy of the left femur was performed to mimic a fracture of the femoral shaft. For the fixation, an intramedullary stainless-steel interlocking nail, a PLA rod or a PLA/PGA rod was used. After 30 months of implantation the histological results of these three materials were examined. In contrast to most reports the degradation rate of both polymers was much lower than the suggested ultimate period of two years. Even the tissue response was more pronounced than expected and this reaction can imply certain risks for repulsion. One can conclude that the volume quantity of polymeric implant in the bony tissue must be reduced if possible to avoid severe foreign body responses. The immunologic responses and the clinical consequences need more studies. The degradation behavior of the polymer is still not under control.

Cortical bone ingrowth in growth hormone-loaded grooved implants with calcium phosphate coatings in goat femurs.

Dental implants are successfully used for tissue-integrated protheses, but the long-term survival in the maxilla is shorter than in the mandible [Cune MS, Thesis, University of Utrecht, 1993; Jaffin RA, Berman CL. J Periodontol 1991;62:2-4]. However, by adding growth hormone at implantation, increased bone apposition may be expected, since it is known that growth hormone has a stimulating effect on the differentiation and proliferation of osteoblastic cells [Ernst M, Froesch ER. Biochem Biophys Res Commun 1988;151:142-47; Scheven BAA et al. Growth Regul 1991;1:160-67; Stracke H et al. Acta Endocrinol 1984;107:16-24]. We studied bone ingrowth and bone contact of grooved implants impregnated with growth hormone in the cortex of femurs of female goats. We compared the effect of growth hormone on grooved implants with or without calcium phosphate coatings at the bottom of the grooves. The coatings used were hydroxyapatite, fluorapatite and heat-treated hydroxyapatite. The implants had both small and large grooves. The implants were positioned in the cortex of one femur and were treated with recombinant human growth hormone, while the implants on the opposite femur served as controls. After 6 weeks, the implants and surrounding tissues were dissected and evaluated histomorphologically and morphometrically by light microscopy. The bone ingrowth and the bone contact in the grooves were quantified by digital image analysis. Calcium phosphate coating at the bottom of the grooves resulted in a significant increase of bone ingrowth and bone contact. Small grooves had significantly more bone ingrowth and bone contact than the larger grooves. However, all implants impregnated with growth hormone showed inhibition of bone contact and bone ingrowth. We conclude that recombinant human growth hormone inhibits bone formation in the grooves coated with calcium phosphate. Without the addition of growth hormone, the calcium phosphate coatings improved bone ingrowth and bone contact in the grooves. Further studies are required to determine whether growth hormone could also possibly act as a bone growth promoting factor in these implants.

Effect of sintering temperature on silica gels and their bone bonding ability.

Silica gels gave rise to apatite precipitation onto their surface depending on the sintering temperature. Silica gels prepared at different sintering temperatures were studied in vivo in cortical bone for their bone bonding ability in relation to their apatite precipitation. From the results we conclude that the sintering temperature influenced the stability of the material. Sintering at the lower temperatures of 400 and 600 degrees C allow the silica gels to be degraded more easily, while gels treated at 900 and 1000 degrees C were more stable. The more stable gels showed some bone bonding, while the degraded gels evoked a high cellular reaction of giant cells and lymphocytes.

In vivo calcium phosphate formation induced by sol-gel-prepared silica.

Implantation with plugs made of a porous sol-gel-prepared silica into the femurs of goats demonstrated that a calcium phosphate was formed both on the silica plugs and within the pores inside the silica plugs 12 weeks postoperatively. This observation indicates that a highly hydrated silica surface is effectively catalytic for calcium phosphate nucleation. Calcification can be triggered in physiologic solution under stimulation of the silica gel. A high level of silicon in the uncalcified osteoid region of young bone is thus thought to provide a number of SiOH groups for initiating calcium phosphate formation. Our results provide some information about the mechanism of calcium phosphate mineralization in higher animals. We believe that heterogeneous nucleation of apatite can be induced from metastable calcium phosphate solutions including physiologic fluids on those specific surfaces of materials, where there are abundant acidic OH groups.

Calcium phosphate plasma-sprayed coatings and their stability: an in vivo study.

Several factors playing a possible role in determining coating stability and bone tissue response were studied in in vivo experiments. These factors involving the plasmaspray coating procedure were as follows: 1) plasmaspray powder port 2 or 6; 2) particle size distribution; 3) hydroxylapatite versus fluorapatite coatings; and 4) the effect of post-heat treatment. Coating stability and bone tissue response were examined by measuring coating thickness, coating length, and bone apposition against the coatings. The result was that heat treatment influenced coating stability significantly. Also, bone formation was more intense. Fluorapatite proved to be more stable than hydroxylapatite, which was in agreement with our previous reports.

Features of calcium phosphate plasma-sprayed coatings: an in vitro study.

Factors involved with the plasma-spray coating procedure, such as starting powder compound (fluorapatite, hydroxylapatite, magnesium-whitlockite, or tetra-calcium phosphate), powder particle distribution 1-45 or 1-125 microns), powder port gun (port 2 or 6), and post-heat treatment of 1 h at 600 degrees C, were examined for their effects on crystallinity and solubility/stability of the coating. From solubility tests, X-ray diffractometry, and scanning microscopy studies, the solubility and crystallinity were found to be dependent on Ca/P ratio, particle distribution, and post-heat treatment. The post-heat treatment influenced the degree of both crystallinity and solubility. The plasma-spray powder port factor for the hydroxylapatite coatings was not significant. Incubation in buffer of the coatings introduced precipitation at the surfaces of all non-heat-treated coatings except fluorapatite. No precipitation could be observed in any of the heat-treated coatings.

Long-term in vivo study of plasma-sprayed coatings on titanium alloys of tetracalcium phosphate, hydroxyapatite and alpha-tricalcium phosphate.

In order to study the interaction of calcium phosphate coatings with bone tissue, coated titanium plugs of standard size were implanted in dog femora. The bone bonding and bone formation of hydroxyapatite, alpha-tricalcium phosphate (alpha-TCP) and tetracalcium phosphate plasma-sprayed coatings were evaluated by mechanical push-out tests and histological observations after 3, 5, 15 and 28 months of implantation. During this time all coating types degraded. alpha-TCP showed the most significant degradation after 3 months of implantation. Hydroxyapatite and tetracalcium phosphate showed significant signs of degradation after about 5 months of implantation. All coatings showed a small increase in bone bonding after 5 months of implantation. In general, all types of implants showed similar bone response, some bone contact and several remodelling lacunae along the surfaces after long-term implantation.

Adherence to a metal, polymer and composite by Staphylococcus aureus and Staphylococcus epidermidis.

Bacterial adherence on to several materials with a potential application in reconstructive surgery was studied. Polymer (poly(L-lactide)), composite (hydroxyapatite/poly(L-lactide)) and metal (316L stainless steel) were evaluated both as smooth and sandblasted specimens. All materials were incubated in phosphate-buffered saline, challenged with Staphylococcus aureus or S. epidermidis and evaluated for up to 24 h. S. aureus showed a preference for the metal and composite tested over the polymer used. For S. epidermidis no preference was found for one of the investigated materials. The influence of surface roughness on bacterial growth was demonstrated by increased colonization on the sandblasted specimens.

Studies of the solubility of different calcium phosphate ceramic particles in vitro.

In vitro solubility tests of hydroxyapatite, tetracalcium phosphate or tricalcium phosphate particles were performed in lactate, citrate, Gomoris or Michaelis buffer with pH 6.2 or 7.2 and in aqua destillata. The results showed that in general the solubility decreased in the order tetracalcium phosphate greater than tricalcium phosphate greater than hydroxyapatite, except for lactate or citrate buffer where the solubility order was tetracalcium phosphate = tricalcium phosphate greater than hydroxyapatite. The influence of the specific buffer used is much larger than either pH or specific calcium phosphate salt tested. The pH stability of lactate buffer and aqua destillata is very low, the other buffer solvents had a rather stable pH value.

Comparison of the hardness change and mineral loss in enamel by dimethyl ammonium fluoride (alpha C12 DMEAHF) or NaF treatment under intra oral cariogenicity test conditions.

Changes in hardness as well as in Ca and P were determined in enamel slabs after treatment with 3% sucrose together with NaF or alpha C12 DMEAHF (both containing 0.006% F-) under Intra Oral Cariogenicity Test conditions. NaF treatment resulted in a hypermineralization of the surface of the enamel lesion suggesting formation of CaF2. Treatment with ammonium fluoride along with the same low F- concentration inhibits the caries process completely and seems to be a very promising anticaries agent.

Hardness changes and mineral loss in enamel during the intra oral cariogenicity test in the presence of 0.125% EHDP with or without 0.1% F-.

The effects of a high EHDP concentration (0.125%) were measured under Intra Oral Cariogenicity Test (ICT) conditions in the presence or absence of F- (0.1%). EHDP as well as EHDP supplemented with F- inhibited the softening of enamel slabs to a similar extent as measured by microhardness. Measurements of the calcium and phosphate levels as a function of depth showed that the addition of F- to the EHDP solution further decreased mineral loss in the deeper layers. The results suggest that the inhibition of demineralization of the enamel by EHDP and F- is due to inhibition of acid production by microorganisms.

Comparison of the effects of 0.1% F- and 0.025% F- on mineral loss in enamel under intra oral cariogenicity test conditions.

Although the caries reducing effect of fluoride (F-) is very well known, the optimal fluoride concentration is still unknown. Therefore we compared the effects of a high (0.1% F-) with a low (0.025% F-) concentration under Intra Oral Cariogenicity Test (ICT) conditions using microhardness measurement and microdissection techniques with which it is possible to quantify the amount of Ca and P present in consecutive enamel layers. No significant differences could be demonstrated between high and low fluoride concentrations in the mineral loss in the surface layer as well as in penetration depth measured perpendicularly to the surface. The reduction of mineral loss after ICT was more pronounced with high fluoride concentrations especially in layers deeper than 150 microns. Using 0.025% F- this effect was restricted more towards the surface. Concomitantly with both F- concentrations a higher Ca/P ratio was observed compared to the sucrose treated or untreated controls.

The effect of daily application of a neutral 0.25% F- mouth-rinse on mineral loss in surface layers of enamel under intra oral cariogenicity tests conditions.

The effects of daily use of a 0.025% F- mouth-rinse on microhardness and mineral loss in the enamel lesion has been studied under Intra Oral Cariogenicity Test (ICT) conditions. The penetration depth was changed from 16 to 9 microns by F-. Moreover, the inhibition was found fairly constantly distributed over the whole lesion for the Ca and P loss without any CaF2 formation. In this way the effectiveness of the 0.025% F- mouth-rinse program can be explained.

Bone mineral content of transilial biopsies in patients with hip fracture.

In a survey of 125 patients with hip fracture vitamin D deficiency was frequently observed, but overt osteomalacia was not found in the bone biopsies (Lips et al., 1982). In order to detect a possible hypomineralization in these vitamin D-deficient patients, we measured the bone mineral content in 64 transilial biopsies, embedded in methylmethacrylate for histomorphometric evaluation. The results were compared with those of 18 bone samples obtained at autopsy from subjects who did not suffer from metabolic bone disease. The calcium:hydroxyproline ratio, the phosphorus:hydroxyproline ratio, and the calcium:phosphorus ratio were similar in the two groups. The magnesium:hydroxyproline ratio was higher in the hip fracture group than in the controls. The ratios did not correlate with serum concentrations of the vitamin D metabolites. The results are not consistent with a decreased bone mineralization in patients with hip fracture.