Towards an intraoperative engineering of osteogenic and vasculogenic grafts from the stromal vascular fraction of human adipose tissue.

Grafts generated by cultivation of progenitor cells from the stromal vascular fraction of human adipose tissue have been proven to have osteogenic and vasculogenic properties in vivo. However, in vitro manufacture of such implants is challenged by complex, impractical and expensive processes, and requires implantation in a separate surgery. This study investigates the feasibility of an intraoperative approach to engineer cell-based bone grafts with tissue harvest, cell isolation, cell seeding onto a scaffold and subsequent implantation within a few hours. Freshly isolated adipose tissue cells from a total of 11 donors, containing variable fractions of mesenchymal and endothelial progenitors, were embedded at different densities in a fibrin hydrogel, which was wrapped around bone substitute materials based on beta-tricalcium phosphate (ChronOS), hydroxyapatite (Engipore), or acellular xenograft (Bio-Oss). The resulting constructs, generated within 3 hours from biopsy harvest, were immediately implanted ectopically in nude mice and analysed after eight weeks. All explants contained blood vessels formed by human endothelial cells, functionally connected to the recipient's vasculature. Human origin cells were also found within osteoid structures, positively immunostained for bone sialoprotein and osteocalcin. However, even with the highest loaded cell densities, no frank bone tissue was detected, independently of the material used. These results provide a proof-of-principle that an intraoperative engineering of autologous cell-based vasculogenic bone substitutes is feasible, but highlight that - in the absence of in vitro commitment--additional cues (e.g., low dose of osteogenic factors or orthotopic environmental conditions) are likely needed to support complete osteoblastic cell differentiation and bone tissue generation.

Comparison between the in vitro surface transformations of AP40 and RKKP bioactive glasses.

Two bioactive silica-phosphate glasses, AP40 and RKKP, were compared in their behaviour in simulated biological environment. Their chemical composition is practically identical, except that RKKP contains small amounts of amphoteric network-former oxides Ta2O5 and La2O3 (composition in wt% for AP40: beta-Ca3(PO4)2 24.50, SiO2 44.30, CaO 18.60, Na2O 4.60, K2O 0.19, MgO 2.82, CaF2 4.99; RKKP: beta-Ca3(PO4)2 24.23, SiO2 43.82, CaO 18.40, Na2O 4.55, K2O 0.19, MgO 2.79, CaF2 4.94, Ta2O5 0.99, La2O3 0.09). Previous investigations showed a better performance in osteopenic bone for RKKP. To gain more insight into these differences in biological behaviour, the in vitro bioactivity of the glasses was studied by treatment with a continuously replenished Hanks' Balanced Salt Solution (HBSS). The glasses were examined before and after HBSS treatment for 20 and 40 days by X-ray Diffraction (XRD), Scanning Electron Microscopy (SEM), X-ray Energy Dispersion (EDX), Raman and IR vibrational spectroscopies. Some slight but notable differences between the two glasses were observed after HBSS treatment. IR and EDX analyses showed that deposits formed on both glasses were composed of a calcium deficient carbonate-apatite; however, the layer formed on RKKP glass was found to be slightly more calcium deficient and thinner. EDX analysis evidenced the presence of a small percentage of F- ions only in the layers formed on the RKKP samples. The differences disclosed, although slight, can contribute to the understanding of the different biological behaviour previously observed.

Physical and chemical aspects of a new porous hydroxyapatite.

The new engineered porous hydroxyapatite (EP-HA) described in this paper seems to be suitable as a bone substitute. Its physico-chemical characteristics and morphology are similar to those of natural bone. Study of its histological behaviour in humans revealed bone formation within and around the porous hydroxyapatite that is markedly resorbed.

Normal osteoconduction and repair in and around submerged highly bisphosphonate-complexed hydroxyapatite implants in rat tibiae.

BACKGROUND: Ceramic hydroxyapatite implants have been used in dentistry for their unique compatibility with alveolar bone. Recently it was reported that bisphosphonates may be beneficial in preventing alveolar bone destruction associated with natural and experimental periodontal disease. Furthermore, bisphosphonate does prevent resorption of alveolar bone following mucoperiosteal flap surgery. We undertook a preliminary study evaluating the effects of highly bisphosphonate-complexed hydroxyapatite implants on osteoconduction and repair in rat tibiae. METHODS: Porous hydroxyapatite implants were pre-incubated in 10(-2)M bisphosphonate solutions at pH 3.49 and pH 7.32. The implants had a diameter of 2.1 mm and a height of 2 mm and adsorbed 115 microg bisphosphonate in vitro. Bisphosphonate/hydroxyapatite implants and plain hydroxyapatite implants were inserted in opposite tibial metaphyses of 35 rats. The measurement errors for the mineral density (MD) of the implants and the proximal trabecular mineral bone density (TD) were estimated by peripheral computed tomography and the bone mineral density (BMD) measurement error by dual x-ray absorptiometry. RESULTS: The measurement errors for the MD of the implants and the TD by peripheral computed tomography were 0.81% and 1.96%, respectively ex vivo. The BMD measurement error estimated by dual x-ray absorptiometry was 0.51% ex vivo. TD and BMD for bisphosphonate/hydroxyapatite implants were insignificantly higher compared to plain hydroxyapatite implants. Bisphosphonate/hydroxyapatite pre-incubated at pH 7.32 were found to be nondegradable implants, while bisphosphonate/hydroxyapatite (pH 3.49) implants were slowly degradable and lost a significant 5% of their density. Histologically, all bisphosphonate/hydroxyapatite implants appeared to be fully integrated and effective as bone replacement material in rat tibial bone. They exhibited vascularization and osteoconduction of tibial bone growth along and inside their porous structure. CONCLUSIONS: Our study suggests that normal osteoconduction and repair occurred in and around the highly bisphosphonate-complexed hydroxyapatite implants in rat tibiae.

Alveolar bone response to submerged bisphosphonate-complexed hydroxyapatite implants.

BACKGROUND: In most studies using submerged hydroxyapatite implants, maintenance of alveolar bone after tooth extraction was attempted with plain hydroxyapatite materials. However, clinical results have shown that hydroxyapatite may require biological modification with a bone resorption-inhibiting agent which may be beneficial for maintenance of alveolar bone. We conducted experimental and clinical studies to evaluate the effect of highly bisphosphonate-complexed hydroxyapatite implants on osteoconduction and repair in alveolar bone. METHODS: Porous hydroxyapatite implants were pre-incubated in 10(-2)M bisphosphonate solutions at pH 3.49. The implants had a diameter of 2.1 mm and a height of 2 mm and adsorbed 115 microg bisphosphonate. Five goats were implanted with 4 plain hydroxyapatite implants on each side of the mandible in root extraction sockets for the precision analysis of dual x-ray absorptiometry (DEXA) measurements. Ten goats were implanted with 4 bisphosphonate/hydroxyapatite implants on one side of the mandible and 4 plain hydroxyapatite implants on the opposite mandible. In a clinical study, 23 bisphosphonate/hydroxyapatite implants were placed in periodontally destroyed tooth root sockets and followed up during one year. RESULTS: The range for the bone mineral density (BMD) measurement errors for goat histologic sections was 0.48% to 1.03%. There were large differences in peri-implant BMDs in the left and right mandible of the same goat, irrespective as to whether hydroxyapatite or bisphosphonate/hydroxyapatite implants were present. This was due to local anatomical differences typical of alveolar bone. These differences were not significant. Histologically, all bisphosphonate/hydroxyapatite as well as hydroxyapatite controls appeared to be fully integrated and effective as bone replacement material in goat alveolar bone. They exhibited vascularization and osteoconduction of alveolar bone growth along and inside their porous structure. In patients peri-implant healing was clinically and radiographically comparable to plain hydroxyapatite implants. All implants were retained and no dehiscences developed. Radiographically, peri-implant radiolucencies disappeared and alveolar bone was deposited in close proximity to the implants. CONCLUSIONS: This study contributes to the understanding of the biological properties of hydroxyapatite implants as carriers for the bone-modulating agent bisphosphonate. Our study suggests that normal osteoconduction and repair occurred in alveolar bone around the highly bisphosphonate-complexed hydroxyapatite implants.

Periodontal membranes from composites of hydroxyapatite and bioresorbable block copolymers.

Biomembranes are frequently proposed as devices for "guided bone regeneration." Such membranes consist generally of a thin sheet of polymeric material, mostly textured from polymeric yarns or clots, which all have a diffuse very fine winding porosity. The cross-section size of the holes of such porosity is nanometric (diameter < 0.1 microm); thus these holes can be indicated as nanoholes. Whatever the method of production, the surface density of nanoholes (number per square centimeter) has to be as high as possible. It is important also that no variation of this density occurs. The fine dimension of these microholes allows the crossing of small molecules (O2, CO2, H2O, sugars, many nutritional organic compounds and even some simple proteins) but not other larger molecules and particulates, including cells of any kind. These biomembranes have, consequently, a semipermeable behavior, providing the functional role which is the interposition of a barrier for the cells, separating the bone from the surrounding soft tissues. The kinetic of proliferation of osteoblasts is lower than that of fibroblasts. Most membranes of this kind are not resorbable. The main problem for the resorbable ones is the speed of size increase of the holes during the time. Their diameter must not exceed a threshold value until the reconstruction of bone is complete, otherwise soft tissue cells will invade the growing bone tissue with formation of undesirable mixed tissue. The present paper deals with a resorbable membrane made with a composite polymer/ceramic. A poly(epsilon-caprolactone)-block-poly(oxyethylene)-block-poly(epsilon-caprolactone) copolymer is the polymeric matrix which contains dispersed ceramic hydroxyapatite microgranules, a stiff filling additive. The main possible use is that of periodontal membranes. The copolymer, obtained by thermal polymerization of epsilon-caprolactone onto poly(ethylene-glycol), presents good biological tolerance, is resorbable under physiological conditions and can promote cell growth. Histological tests, performed 6 months after implantation, showed that the polymeric matrix is almost totally resorbed. New-formed bone colonizes even the innermost parts of the membrane, with bone trabeculae closely surrounding the hydroxyapatite granules.

Resorption of composite polymer-hydroxyapatite membranes: a time-course study in rabbit.

The histological findings in rabbit, concerning the resorption of a new composite hydroxyapatite-polymer membrane used in guided tissue regeneration are reported. The polymer appeared to be completely resorbed in about 4-6 months, while the inorganic component, hydroxyapatite, appeared to begin to resorb after the tenth month. The integrity of the membrane appeared to be unchanged after a 10-month period. No inflammatory cell infiltrate was present. This new type of membrane appeared to be relatively well tolerated by the host, had no significant adverse soft and hard tissue reaction, appeared to be easy to handle and had good space-maintaining capabilities. More research is certainly needed before clinical use in man.

[Necessity and validity of standard models for experimental preclinical evaluation of biomaterials. An example of biologic characterization of a hydroxyapatite-based implant material]. / Necessità e validità di modelli standard per la valutazione sperimentale preclinica dei biomateriali. Un esempio di caratterizzazione biologica di un materiale da impianto a base di idrossiapatite.

A large number of methods are now available for the preclinical screening of implantable materials concerning their biocompatibility and their ability to stimulate tissue formation. In vitro techniques represent a very useful tool, since this way we can realistically simulate the biological events which occur in vivo at the bone-implant interface. In the present study scanning electron microscopy and light microscopy observations were performed in order to assess the effect of an hydroxyapatite granulate on cell behaviour and morphology. Uptake of proteins to hydroxyapatite surface has been also investigated by comparing the amounts adsorbed after incubation with bovine serum albumin and bovine pancreaticamilase. According to our preliminary observation cells do not show signs of toxicity or inhibition of cell growth even after 14 days of co-culture with hydroxyapatite. Granules were covered by an uninterrupted cell layer by day seven. Even after two days micrographs show cells anchored and spread over the surface of the underlying granules, with a flattened and stellate shape. Such a morphology indicates a very high cellular activity, suggesting that the interaction with hydroxyapatite seriously increased metabolism. Measurements of protein adsorption on the hydroxyapatite surface show that changes in the size of particles affect the binding of proteins, while, in the case of granular hydroxyapatite, despite changes in size of granules, variations of protein adsorption were not observed, neither in relation to their different isoelectric point. Our preliminary results represent a good example of the opportunities presented by an experimental in vitro model.

Net-shaped hydroxyapatite implants for release of agents modulating periodontal-like tissues.

Periodontal-like tissues and, in particular, alveolar bone- and root cementum-like material can theoretically be modulated by release of biochemical agents such as bisphosphonate (PCP), growth hormone (GH) and alkaline phosphatase (ALP) from the implant surface. The present research focused on porous ceramic hydroxyapatite (PCHA) implants. In the past the PCHA implants were machined on a lathe out of simple blocks of PCHA ceramic. This was a tedious and cumbersome method, often resulting in implants with undesirable characteristics: different porosities, cracks and fractures. Therefore a moulding technique was developed to sinter near-net-shaped PCHA implants at 2 different sintering temperatures: 1170 degrees C and 1280 degrees C, resulting in PCHA implants with porosities of 62.06% (PCHA type 1) and 40.74% (PCHA type 2), respectively. After 1 h incubation in a 10(-2) M solution of PCP, the total amounts adsorbed onto PCHA type 1 and type 2 were 114.9 +/- 2.1 micrograms and 46.1 +/- 1.5 micrograms, respectively. This was approximately 5 times higher than after incubation for 1 wk in a 10(-4) M solution of PCP. The total amounts of PCP released after the observation period of 75 d from PCHA type 1 and type 2 after incubation in the 10(-2) M solution were 103.1 +/- 1.8 micrograms and 42.8 +/- 1.5 micrograms, respectively. The total amounts released from type 1 and 2 after incubation in the 10(-4) M solution were 7.4 +/- 0.4 micrograms and 4.1 +/- 0.1 micrograms, respectively. After 2 wk of incubation in a liver/bone/kidney ALP solution the total amount of ALP adsorbed onto PCHA type 1 implants was 5039 +/- 412 mU/ml. The total amounts of ALP released were 4674 +/- 438 mU/ml and 53 +/- 20 mU/ml after 1 and 2 wk, respectively. The release of ALP was high at the beginning but slowed down thereafter. It was evident that despite the well-known high bonding affinity of PCP to HA the release of PCP occurred steadily, over a long period of time in vitro.

Interface between hydroxyapatite and mandibular human bone tissue.

Samples from intraosseous dental implants, removed from patients for mechanical failures, were examined to analyse the interaction between hydroxyapatite as plasma sprayed coating on titanium supports and human bone. The implantation time varied up to 8 years. No failures had arisen from problems at the interface between the hydroxyapatite coating and bone. The number of samples examined and the implantation times give good statistical conclusions. Histological and microchemical studies showed the good performance and compatibility of this sprayed hydroxyapatite. We present evidence from the best samples which show close bonding with the surrounding bone tissue. New bone is seen all around the coated implant. The composition of the calcium phosphate deposited on the hydroxyapatite and cellular approach were determined, and demonstrate the efficiency of the interaction between this plasma sprayed hydroxyapatite and the bone.

Cognitive antecedents of dream recall.

222 students completed the Cognitive Processes Survey which assessed imaginal life, orientation toward imaginal life, and defensiveness. Subjects were separated according to number of weekly dreams recalled and tested for short-term memory with the Digit Span of the Wechsler Adult Intelligence Scale. Analyses of variance showed that imaginal life differed significantly across low, average, and high dream recallers. Orientation toward imaginal life was significant for high dream recallers but not for low recallers. A t test for correlated Digit Span raw scores indicated significant differences between low and high dream recallers. Differences in dream recall seemed better explained by cognitive variables such as short-term memory than attitudinal factors such as defensiveness. Dream recall might be enhanced by increasing the channel capacity of short-term memory and increasing imaginal life through activities such as introspection, daydreaming, and meditation.

Effect of stimulus perspective on perception of oscillation.

8 groups of subjects, 10 per group, had 10 trials for each of two perceptual tasks. Subjects in each group viewed a rotating trapezoid at a constant speed at equal-interval angles of tilt (0 degrees, 30 degrees, 60 degrees, 90 degrees) and two directions of stimulus tilt (toward observer and parallel to observer). It was predicted that most reports of oscillatory perception would occur for parallel tilt at 0 degree and 90 degree angles of orientation. In addition, there would be a hypothesized decrease in perceived oscillations from 0 degrees to 90 degrees when the stimulus was tilted toward the observer. Both direction of stimulus tilt and angle of orientation were significant. Post hoc comparisons yielded reliable differences from the parallel tilt and tilt toward the observer. The evidence supports the position that veridical information processing in the trapezoid oscillatory illusion is a joint function of signal-detection capability and availability of perceptual cues.

Intersubject variability in perceived oscillation as a function of threshold for binocular disparity.

Four groups of 15 female subjects each were classified along a quantitative dimension for proportion of perceived oscillation and threshold for binocular disparity. Analyses of variance showed that significant differences in proportion of perceived oscillation were accompanied by significant differences in threshold for binocular disparity for perceivers of "high" and "low" oscillation (p less than .05). For perceivers of "intermediate" oscillation significant differences in proportion of perceived oscillation but not threshold for binocular disparity were found. It was suggested that: (1) intersubject variability in perceived oscillation may be governed by the threshold for binocular disparity, (2) "low" perceivers may be especially sensitive to the magnitude of the cue, (3) "intermediate" perceivers' subjective reports may be primarily dependent on response criteria and the multiplicity of subjective factors which constitute it, (4) "high" perceivers apparently have least response sensitivity and they cannot maintain a consistent response criterion.

The effect of visual angle orientation on perceived oscillatory motion.

The present experiment examined the effect of independently varying central, vertical, and horizontal visual angles (CVVA, CHVA) on proportion of perceived oscillations with rotating trapezoids. Twenty-five female undergraduates served as Ss. Diminution of the CHVA proved most effective in increasing oscillatory perception (p less than .01), although perceived oscillation was found to be inversely proportional to CVVA and CHVA (p less than .001). The data indicate that Os primarily rely on perceptual cues determined by the horizontal dimension of a trapezoid. When cue discriminability is reduced, the results suggest that O's perceptual response may be a function on individual differences in cue thresholds, and/or response criteria.

The role of perceptual cues in the trapezoid oscillatory illusion.

The present experiment was designed to test the hypothesis that binocular disparity functions as a veridical cue, and linear perspective as a nonveridical cue in the perception of a rotating trapezoid. Three trapezoids with proportionately increasing slope were viewed by 10 subjects through viewing screens designed to control the availability of binocular disparity. Monocular and binocular viewing conditions were employed. The dependent variable, proportion of perceived oscillation, was analyzed by a factorial analysis of variance and appropriate post hoc tests. The obtained data indicate that increasing the availability of binocular disparity by increasing viewing screen width favors veridical rotation perception. When the binocular depth cue was attenuated by decreasing viewing screen width, the observer relied on linear perspective, and non-veridical oscillation perception was favored. As linear perspective was increased by increasing trapezoid slope, perceived oscillation increased both monocularly and binocularly. However, for any level of linear perspective, monocular viewing invariabily produced more oscillation than binocular viewing, indicating that monocular depth cues (movement parallax and interposition) were not as effective at neutralizing the illusory perspective effect as the binocular disparity cue.