Effects of sterilization and storage on the properties of ALP-grafted biomaterials for prosthetic and bone tissue engineering applications.

Grafting of the biomaterial surfaces with biomolecules is nowadays a challenging research field for prosthetic and bone tissue engineering applications. On the other hand, very few research works investigate the effect of the sterilization processes on the properties of functionalized biomaterials. In this study, the effects of different sterilization techniques (e.g. gamma and electron beam irradiation, ethylene oxide) on the enzymatic activity of bioactive glasses and Ti6Al4V grafted with alkaline phosphatase (ALP) have been analyzed. Sterility maintenance and in vitro bioactivity of the sterilized surfaces have also been investigated. Finally the effect of packaging and storage conditions has been considered.

Treatment of recalcitrant ulcers with allogeneic platelet gel from pooled platelets in aged hypomobile patients.

We evaluated growth factor contents and clinical efficacy of allogeneic platelet gel (PG) prepared with standard blood banking procedures from routine platelet concentrates (PCs) obtained from buffy coats. The PGs were used to treat 11 hypomobile very elderly patients unable to undergo autologous blood processing and previously ineffectively treated with expensive advanced medications for 8-275 weeks. PGs were prepared by platelet activation with human thrombin or commercial batroxobin. Median and range growth factor contents (ng/mL) were: platelet derived growth factor (PDGF-AB/-BB) 112 (31-157) and 20 (3.8-34); transforming growth factor (TGF-ß1/-ß2) 214 (48-289) and 0.087 (0.03-0.28); basic-fibroblast growth factor (b-FGF) 0.03 (0.006-0.214); vascular endothelial growth factor (VEGF) 1.15 (0.18-2.46); epidermal growth factor (EGF) 4.50 (0.87-6.64); insulin-like growth factor (IGF-l) 116 (72-156). In the clinical study, 222 PGs were used within 2 h of activation to treat 14 chronic skin ulcers in the 11 patients. No improvement was seen in 3 patients with 24, 27 and 30 cm(3) ulcers who could be treated for no more than 4, 7 and 8 weeks due to progressively worsening clinical conditions, while 11 ulcers with 3.2 cm(3) median size (range 0.2-3.6) in the remaining 8 patients showed 91 ± 14 % reduction after a median of 12 weeks (range 1-20). Cost of PG treatment (19,976 euro) amounted to about 10% of the ineffective advanced medication hospital reimbursement fees (191,236 euro). This study supports efficacy and feasibility of allogeneic PG to treat recalcitrant ulcers in very elderly hypomobile patients for whom autologous blood processing may be difficult.

Not every PRP-gel is born equal. Evaluation of growth factor availability for tissues through four PRP-gel preparations: Fibrinet, RegenPRP-Kit, Plateltex and one manual procedure.

BACKGROUND: The rationale for using topical platelet gel therapy is to provide the healing tissues with concentrated platelet-derived factors. Several systems are available to prepare platelet-rich plasma (PRP) and from these, the platelet gel. These systems produce two- to six-fold platelet and growth factor-enriched concentrations. The bioavailability of growth factors in tissue healing depends on the amount of growth factors stored in platelets but a portion of these is lost during platelet manipulation. Very few data have been reported on the kinetics of growth factor release from PRP-gels. The aim of this study is to assess the growth factor recovery and its bioavailability to tissues in four different PRP and PRP-gel preparation techniques. MATERIALS AND METHODS: Three commercially available devices (Fibrinet, RegenPRP-Kit, Plateltex) and one manual procedure (home made, HM) were evaluated with reference to resulting platelet concentration, growth factor content and the kinetics of growth factor release from gel. RESULTS: Platelet concentration increased from 1.65- to 4.4-fold in comparison with whole blood initially used. The final platelet concentration (x 10(3)/microl) was: Fibrinet 1358 +/- 419, Regen 430 +/- 109, HM 1196 +/- 188, and Plateltex 1160 +/- 164. A high variation (5- to 27-fold) was found in growth factor concentration in relation to the method used and also a high variation in the kinetics of growth factor release from gels. CONCLUSIONS: Similar methods for platelet gel preparation revealed different performances concerning growth factor recovery and the kinetics of its release from the gel. It is unclear whether these noticeable differences are important for clinical management.

Platelet lysate stimulates wound repair of HaCaT keratinocytes.

BACKGROUND: Platelets play a pivotal role in wound healing. Their beneficial effect is attributed to the release of bioactive substances, although the involved mechanisms are mostly unknown. OBJECTIVES: To investigate mechanisms underlying platelet-induced wound healing using HaCaT keratinocytes, representing an in vitro model of proliferating and migrating keratinocytes. METHODS: Cells were exposed to platelet lysate (PL) purified from whole blood samples. Cell metabolism and proliferation were assessed using MTS and crystal violet assays, respectively, wound healing was assessed by scratch wound assay and cell migration by transwell assay. Extracellular signal-regulated kinase (ERK) 1/2 and p38 activations were studied using Western immunoblotting and intracellular Ca(2+) dynamics by confocal imaging. RESULTS: Wound closure rates showed a significant increase at 6 and 24 h in cells exposed to nontoxic 20% PL. The cell migration assay showed a strong chemotactic effect toward PL. The intracellular Ca(2+) chelator BAPTA-AM induced 100% inhibition of the PL effect on wound closure rate, while among the kinase inhibitors, SB203580 exerted about 50% inhibition, and PD98059, wortmannin and LY294002 about 30% inhibition. SB203580 and BAPTA-AM induced 100% inhibition of the PL effect on cell migration, PD98059 about 50% inhibition, and wortmannin and LY294002 no significant inhibition. Confocal imaging allowed detection of a sustained Ca(2+) transient in PL-treated cells, while Western blot showed a more rapid activation of p38 than of ERK1/2. CONCLUSIONS: Data indicate that PL increases wound healing rate by stimulating keratinocyte migration through a calcium- and p38-dependent mechanism. ERK1/2 and phosphoinositide-3 kinase seem to play minor roles.

Platelet-rich plasma and platelet gel preparation using Plateltex.

BACKGROUND: The platelet gel is made by embedding concentrate platelets within a semisolid (gel) network of polymerized fibrin. It is believed that this blood component will be used more and more in the treatment of several clinical conditions and as an adjunctive material in tissue engineering. Several systems are available to produce platelet-rich plasma (PRP) for topical therapy. Recently, a new system became commercially available, Plateltex. Here we report the technical performance of this system in comparison with the performance of other commercially available systems: PRGF, PRP-Landesber, Curasan, PCCS, Harvest, Vivostat, Regen and Fibrinet. MATERIAL AND METHODS: Both the PRP and the gel were prepared according to the manufacturer's directions. The blood samples of 20 donors were used. The yield, the efficiency, and the amount of platelet-derived growth factor AB (PDGF-AB), transforming growth factor beta, vascular endothelial growth factor and fibroblast growth factor were measured in the resulting PRP. The feature of the batroxobin-induced gelation was evaluated. RESULTS: The yield, the collection efficiency and the growth factor content of Plateltex were comparable to those of most of the other available systems. The gelation time was not dependent on the fibrinogen concentration; however, it was strongly influenced by the contact surface area of the container where the clotting reaction took place (P < 0.0001). CONCLUSIONS: Plateltex provided platelet recovery, collection efficiency and PDGF-AB availability close to those provided by other systems marketed with the same intended use. Batroxobin, the enzyme provided to induce gelation, acts differently from thrombin, which is used by most other systems. Platelets treated with thrombin become activated; they release their growth factors quickly. Furthermore, thrombin-platelet interaction is a physiological mechanism that hastens the clot-retraction rate. On the contrary, platelets treated with batroxobin do not become activated; they are passively entrapped within the fibrin network, and their growth factor release occurs slowly. In these conditions, the clot retraction takes longer to occur. According to these differences between thrombin and batroxobin, it is expected that batroxobin-induced PRP activation will tailor slow release of the platelet content, thus, providing longer in loco availability of trophic factors. In selected clinical conditions, this durable anabolic factor availability might be preferable to quick thrombin-induced growth factor release.

Collagen I-coated titanium surfaces: mesenchymal cell adhesion and in vivo evaluation in trabecular bone implants.

The goal of the study was the evaluation of the effect of modification of titanium implants by acrylic acid surface grafting-collagen I coupling. Tests were performed on titanium samples treated by galvanostatic anodization to create a porous surface topography. Surface characterization by X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM) confirms the biochemical modification of the surface and shows a surface topography characterized by pores mostly below 1 mum diameter. In vitro evaluation involving human mesenchymal cells shows enhanced cell growth on collagen coated surfaces as compared to titanium ones. Four weeks in vivo evaluation of implants in rabbit femur trabecular bone shows improvements of bone-to-implant contact, while improvement of bone ingrowth is slightly not significant (p = 0.056), when compared to the control. Overall, these data indicate that integration in trabecular, or cancellous, bone can be enhanced by the surface collagen layer, confirming previous findings obtained by modification of machined surfaces by the same approach in cortical bone implants.

Enhanced osmotolerance of a wheat mutant selected for potassium accumulation.

A mutant of durum wheat was identified by screening a M4 population (sodium azide) for genotypes with enhanced capacity for potassium accumulation in leaves. The mutant (designated 422) was grown in field, controlled environment, hydroponic culture and NaCl salinized soil. Mutant 422 accumulates about 5 mg/g dry weight more K than the wild-type and is less salt sensitive, based on leaf growth and germination. During vegetative growth exists a specific tolerance of the 422 mutant to K(+) ion and a moderate tolerance to Cl(-) ion, in hydroponic culture. Under severe stress imposed by salts and mannitol, the mutant germinates better than wild type (WT). In soil containing increasing NaCl, mutant 422 had higher potassium amount than WT, but did not show augmented capacity to concentrate the ion in the leaves as salt stress increased. The capability to accumulate potassium could improve tissue hydration, because water content of 422 leaves was greater than WT and increased linearly in relation to leaf K(+) concentration.

Evaluation of the hemostatic function of stored platelet concentrates using the platelet function analyzer (PFA-100 ).

BACKGROUND AND OBJECTIVE: Progressive functional impairment is known to occur in platelet concentrates through the storage period. Standardized methods providing direct measurement of residual platelet function in stored platelets are lacking. The purpose of this study was to determine whether a new platelet function analyzer (PFA-100 ) could provide standardized methods for assessing the hemostatic capacity of stored platelets. DESIGN AND METHODS: The PFA-100 was used to evaluate platelet function in stored platelets. The instrument can process citrated whole blood but it is unable to process platelet suspensions. Accordingly, the function of platelet concentrates should be measured following reconstitution of pseudo-whole blood. The analysis of the results included the closure time (sec) and a predictive index, an arithmetical index computed on the basis of the instrument's output data: the flow rate, the flow volume, the closure time. RESULTS: A final hematocrit of 58+/-2 and a final platelet concentration of 230+/-20x10(9)/L were used as standardized operative conditions to measure the function of stored platelet concentrates. The closure time (PFA-CT) and the predictive index (PFA-PI) both resulted to be capable of discriminating platelet concentrates with maintained or impaired function. PFA-PI was more informative than PFA-CT in terms of description of the residual platelet function. Of the two agonists used, epinephrine (EPI) resulted to be particularly sensitive for the detection of initial platelet hyporeactivity, whereas adenosine 5'-diphosphate (ADP) was particularly useful for measuring the residual platelet reactivity. INTERPRETATION AND CONCLUSIONS: PFA-CT and PFA-PI can be standardized; they provide new information about the hemostatic function of stored platelet concentrates and can be used to assess the quality of platelet concentrates.