Assessment of organ culture for the conservation of human skin allografts

Objective Human skin allografts are used in the treatment of severe burns and their preservation is therefore critical for optimal clinical benefit. Current preservation methods, such as 4°C storage or cryopreservation, cannot prevent the decrease of tissue viability. The aim of this study was to assess viability and function of skin allografts in a new skin organ culture model, allowing conservation parameters as close as possible to physiological conditions: 32°C, air–liquid interface and physiological skin tension. Design Twelve skin samples, harvested from 6 living surgical donors, were conserved 35 days in two conditions: conservation at 4°C and organ culture. Viability and function of skin samples were investigated at Day 0, 7, 14, 21, 28 and 35 using cell culture methods (trypan blue exclusion, Colony Forming Efficiency and Growth Rate), histopathological and histoenzymological studies (Ki67 immunostaining). Results In the two conditions, fibroblast and keratinocyte viability was progressively affected by storage, with a significant decrease observed after 35 days. No statistical difference could be observed between the two conditions. The two methods were also comparable regarding alterations of fibroblast and keratinocyte culture parameters, which were respectively significantly reduced at Day 7 and 21, compared to fresh skin. By contrast, histopathological and histoenzymological studies revealed a better preservation of skin architecture and proliferative potential at 4°C, as compared to organ culture. Conclusion These results indicate that skin organ culture does not provide significant advantages for skin allograft preservation. However, its potential use as an experimental model to study skin physiology and wound healing should be further evaluated.     

Effects of storage temperature on viable bioprosthetic heart valves.

Long-term in vivo success of bioprosthetic allografts is dependent upon retention of cellular functions, such as protein synthesis. The purpose of the experiments presented in this report was to determine the storage conditions necessary for retention of protein synthetic functions in human allograft heart valve leaflets. Tissue viability was assessed by measurement of tritiated-glycine incorporation into proteins. Comparison of short-term (less than 3 month)- and long-term (1 and 2 years)-cryopreserved heart valve leaflet storage in a liquid nitrogen freezer below -135 degrees C demonstrated preservation of fibroblast protein synthesis. In contrast, storage in a mechanical freezer at -80 degrees C resulted in a time-dependent loss of fibroblast protein synthesis. There was no statistically significant effect on protein synthesis in leaflets stored for 1 week at 4 degrees C compared to control cryopreserved liquid nitrogen-stored leaflets. After 2 weeks of 4 degrees C storage leaflet protein synthesis declined significantly to 15% that of cryopreserved controls. These results demonstrate that liquid nitrogen storage of valve bioprostheses is required for long-term preservation of cellular functions.     

Differences in preservation of canine chilled semen using different transport containers

In the present study, the effect of three different containers in the preservation of dog chilled semen, during 24, 48 and 72h was evaluated. Weekly sperm pools of different dogs were obtained, during 10 consecutive weeks. Semen samples were diluted in egg-yolk-Tris-fructose extender and stored in a Styrofoam box, a common Thermos flask and an Equitainer. Progressive motility, morphology and sperm membrane integrity were examined in semen aliquots taken daily from each container during the 3 days of storage. Additionally, integrity of the acrosome and sperm plasma membranes, determined by PI/Fitc-PSA staining was assessed at 48 and 72h of storage. At 24h no differences were observed between the three containers for the evaluated parameters. At 48h samples kept in the Equitainer presented a higher progressive motility than samples kept in the Thermos. At 72h, progressive motility was higher in the Equitainer than in the other two containers. Only samples kept in the Equitainer maintained similar levels of progressive motility between 24 and 72h. Membrane integrity assessed by eosin-nigrosin deteriorated over the 72h period, whereas functional membrane integrity determined by the hypoosmotic swelling test was independently affected by type of container (the Equitainer) kept a higher percentage of sperm cells with intact membrane) and time of storage (a decrease of membrane integrity between 24 to 72h). Staining with PI-Fitc-PSA allowed the detection of differences between containers but not between the two studied storage periods (48 and 72h). The results indicated that the use of the Equitainer is preferable when transporting chilled dog semen for more than 48h.     

Influence of freezing with liquid nitrogen on whole-knee joint grafts and protection of cartilage from cryoinjury in rabbits

Improving survival rates for sarcoma patients are necessitating more functional and durable methods of reconstruction after tumor resection. Frozen osteoarticular grafts are utilized for joint reconstruction, but the joint may develop osteoarthritic change. We used a frozen autologous whole-rabbit knee joint graft model to investigate the influence of freezing on joint components. Thirty rabbit knee joints that had been directly immersed into liquid nitrogen (L) or saline (C) without use of cryoprotectants were re-implanted. Histological observations were made after 4, 8, and 12 weeks. Both groups had bone healing. In group L, despite restoration of cellularity to the menisci and ligaments, no live chondrocytes were observed and cartilage deterioration progressed over time. It was concluded that cryoinjury of chondrocytes caused osteoarthritic change. Then we tested whether a vitrification method could protect cartilage from cryoinjury. Full-thickness articular cartilage of rabbit knee was immersed into liquid nitrogen with and without vitrification. Histology, ultrastructure, and chondrocyte viability were examined before and after 24 h of culture. Vitrified cartilage cell viability was >85% compared with that of fresh cartilage. Transmission electron microscopy revealed preservation of original chondrocyte structure. Our vitrification method was effective for protecting chondrocytes from cryoinjury. Since reconstructing joints with osteoarticular grafts containing living cartilage avert osteoarthritic changes, vitrification method may be useful for storage of living cartilage for allografts or, in Asian countries, for reconstruction with frozen autografts containing tumors.     

Low-cost and low maintenance preservation of Agaricus brasiliensis cultures

Agaricus brasiliensis cultures quickly lose viability when stored at cool temperatures, even for a short period of time. We evaluated several low-cost preservation methods using varied substrates, preservation solutions, and storage temperatures. Agaricus brasiliensis was intolerant to freezing temperatures, making liquid nitrogen use and deep-freezing methods impossible for its preservation. The best preservation conditions for the A. brasiliensis CS1 strain tested in this study were obtained by using rice as substrate and water as preservation solution, with storage at room temperature or when using soil, mushroom cultivation compost, or rice and stored at 10 °C without preservation solution. Those cultures that were reactivated showed the same productivity attributes as the control. In addition, no effect on productivity or biological efficiency was observed through successive subculturing of the strain (CS1). Parboiled rice was successfully used for other A. brasiliensis strains (CS2, CS5, CS7, CS9, and CS10), and also for Pleurotus ostreatus, P. sajor-caju, and Lentinula edodes.     

Design and management of an orthopaedic bone bank in the Netherlands

The design and management of an orthopaedic bone bank is a complex process in which medical organisation and legislation intertwine. Neither in the Netherlands, nor in any other European country, there are official guidelines for the organisation and management of an orthopaedic bone bank. In the Netherlands, the recently modified ‘law of security and quality for using human materials’ (WVKL) dictates requirements for technical and organisational aspects for the use of human tissue and cells. The bone bank procedures include a thorough questionnaire for donor selection, extensive serological, bacteriological and histopathological examination, as well as standard procedures for registration, processing, preservation, storage and distribution of bone allografts. This article describes the organisation of an accredited bone bank and can be used as a proposition for an official guideline or can be useful as an example for other orthopaedic bone banks in Europe.     

Differential patterns of incorporation and remodeling among various types of bone grafts

The early (3 months) and later (6 months) patterns of incorporation and bone formation have been evaluated histomorphometrically for different types of bone grafts; that is, vascularized and nonvascularized autografts with and without ciclosporin, and vascularized and nonvascularized dog leukocyte antigen (DLA)-mismatched allografts with and without ciclosporin. The vascularized bones were superior to the nonvascularized ones in healing and remodeling their grafted segments. In the autograft bones, ciclosporin did not alter the incorporation process 3 months after transplantation but delayed and increased the remodeling activities in the long run (6 months). Nonvascularized allografts underwent vigorous resorption, and were markedly porotic. Ciclosporin administration significantly reduced resorption and enhanced remodeling in nonvascularized allografts. The remodeling of allografts was similar to that of autografts in the presence of ciclosporin, but stopped soon after the administration of ciclosporin ceased.     

Lung transplantation protection after warm ischemia and 1 day preservation

This study attemps to determine the role of colloid hyperosmolar solutions in the preservation of nonischemic and ischemic lungs. Four groups of animals were studied: Control fresh lung allografts and lungs stored under hypothermic storage (4–7 °C) in a modified silica gel fraction (MSGF) for 24 hr with or without warm ischemia (0, 30, 60 min) prior to storage. Fresh lungs lived an average of 14.5 days after transplantation. Stored nonischemic lungs survived an average of 11.2 days, and ischemic (30, 60 min) stored lungs remained alive for an average of 10.5 and 9.2 days. There were no significant differences in survival between the fresh and preserved allografts. Pneumonia and/or rejection occurred in 71% of all groups. MSGF appears to be a good solution for 24-hr hypothermic storage of nonischemic and ischemic lungs.     

Productivity of Agaricus brunnescens stock cultures following 5-, 7-, and 10-year storage periods in liquid nitrogen

Eight cultivars of the commercial mushroom, A. brunnescens, were preserved in liquid nitrogen for 10 years with no apparent changes in morphological or physiological characteristics. With one exception, cropping trials after 5-, 7-, and 10-years storage showed no significant differences in the productive capacity of frozen cultures as compared to the same cultivars maintained by conventional mycelial transfer. Cryogenic preservation of mushroom stock cultures was shown to be a realistic, practical method of long-term conservation. The major advantages of nitrogen storage are the preservation of the original genetic lines, protection of cultures from loss through contamination or mutation, and the reduction of time, materials, labor and space necessary for conventional maintenance.     

Effects of transport container and ambient storage temperature on motion characteristics of equine spermatozoa

This study was conducted to compare the cooling rates and storage temperatures within equine semen transport containers exposed to different ambient temperatures, and to evaluate the ability of these containers to preserve spermatozoal motility following 24 h of storage under these conditions. In Experiment 1, nonfat dried milk solids, glucose, sucrose, equine semen extender was divided into seven 40-mL aliquots and loaded into seven different semen transport containers: Equitainer I, Equitainer II, Equitainer III, ExpectaFoal, Bio-Flite, Lane STS, and Equine Express. After containers were loaded, they were subjected to one of three ambient storage temperatures: 1) 22 degrees C for 72 h, 2) -20 degrees C for 6 h followed by 22 degrees C for 66 h, or 3) 37 degrees C for 72 h. Cooling rates and storage temperatures of semen extender in each container were monitored with thermocouples and a chart recorder. In Experiment 2, semen from each of three stallions (3 ejaculates per stallion) was diluted to 25 x 10(6) spermatozoa/mL with semen extender, divided into 40 mL aliquots and loaded into transport containers as in Experiment I. Containers were subjected to one of three ambient storage conditions: 1) 22 degrees C for 24 h, 2) -20 degrees C for 6 h, followed by 22 degrees C for 18 h, or 3) 37 degrees C for 24 h. After 24 h of storage, spermatozoal motion characteristics (percentage of motile spermatozoa; MOT, percentage of progressively motile spermatozoa; PMOT, and mean curvilinear velocity; VCL) were evaluated using a computerized spermatozoal motion analyzer. Significant interactions were detected among storage conditions and semen transport containers for the majority of the temperature endpoints measured. When exposed to temporary ambient freezing conditions, the lowest temperatures attained by samples in containers ranged from -2.8 to 0.8 degrees C. Lowest temperature samples attained was not correlated (P > 0.05) with spermatozoal motility under any ambient condition. However, time below 4 degrees C was highly correlated (P < 0.05) with a reduction in spermatozoal motility. Mean cooling rates from 20 degrees C to 8 degrees C did not correlate with spermatozoal motility, except when containers were exposed to temporary freezing conditions. No container cooled samples below 6 degrees C in 22 degrees C or 37 degrees C environments except for the ExpectaFoal, in which samples fell below 4 degrees C under all ambient conditions. Ambient temperature affected MOT, PMOT and VCL of semen stored in all containers (P < 0.05) except for the Equitainer II in which motion characteristics remained high and were similar among all ambient temperatures (P > 0.05). Results suggest that stallion semen may be able to tolerate a wider range of cooling rates and storage temperatures than previously considered safe.     

Vascularized bone allografts: in vitro assessment of cell-mediated and humoral responses

The immunologic consequences of transplantation of vascularized bone allografts have not been previously characterized. In this study, knee allografts, both vascularized and nonvascularized, were transplanted from Lewis rats to Brown Norway rats across a strong histocompatibility barrier. A total of 66 transplants and 8 control animals were evaluated. The vascularized knee grafts consisted of 1 cm of proximal tibia and distal femur with a minimal muscular cuff isolated on the femoral vessels, and these were transplanted to a heterotopic, subcutaneous position on the abdominal wall of the recipient rat. Nonvascularized allografts (identical but without anastomoses) were transplanted for comparison. The cell-mediated response was measured by lymphocytotoxicity assay, and the humoral response was measured by cytotoxic antibody assay, both employing 51Cr-labeled target cells. The timing and intensity of the immune response differed according to the type of graft. The vascularized bone allografts generated significant cell-mediated and humoral responses as early as 5 days posttransplant. A significant humoral response in nonvascularized bone allografts was not apparent until day 14, while cell-mediated response in these grafts was variable. These findings were correlated with the histologic appearance of the grafted tissue. Cyclosporine, which was administered to one group of vascularized bone allografts, resulted in the suppression of both types of immune responses. The histologic appearance of this group resembled that of isografts transplanted as controls. The clinical application of vascularized bone allografts may offer significant advantages over nonvascularized allografts in the reconstruction of massive bone defects. Complications such as nonunion, fracture, and collapse of articular segments seen in nonvascularized allograft transplantation may be avoided by preservation of the blood supply to the graft. Characterization of the immune response to vascularized bone allografts may subsequently allow the manipulation of the host and/or graft tissue and promote graft incorporation.     

Immunologic and ultrastructural changes during early rejection of vascularized bone allografts

This investigation evaluated ultrastructural changes during the earliest phase of immunologic rejection of vascularized bone allografts in a genetically defined rat model. These results were correlated with the cell-mediated and humoral immunologic responses during this time period. Employing a model for heterotopic allograft transplantation, 33 rats divided into four categories were evaluated. Group I consisted of ungrafted (naive) Lewis and Brown Norway rats; group II consisted of Lewis-to-Lewis vascularized bone isografts; group III consisted of Lewis-to-Brown Norway vascularized bone allografts; and group IV consisted of Lewis-to-Brown Norway vascularized bone allografts in rats receiving cyclosporine (10 mg/kg/day). Experimental animals were sacrificed at 3, 5, and 7 days. Immunologic analysis was performed using a cell-mediated lymphocytotoxicity assay and a complement-dependent cytotoxic antibody assay. The results of this study show that rejection of vascularized bone allografts appears as early as 3 days postoperatively, with osteocytes and vascular endothelium being the first elements affected. This early rejection is probably a manifestation of the humoral response. All changes secondary to rejection were arrested by cyclosporine.     

The Effects of Prolonged Cryopreservation on the Biomechanical Properties of Bone Allografts: A Microbiological, Histological and Mechanical Study

Bone allografting is the most common form of allotransplantation in modern medicine. Bone banking is usually the major part of most tissue banks throughout the world. Several years ago, many standards of bone banking were set empirically, and have never been evaluated. One particular parameter or standard was outdating graft materials after 5 years of storage. This study was conducted to evaluate the effect of prolonged cryopreservation on the biomechanical properties of bone allografts and establish whether graft materials become contaminated during long-term storage.Proximal humeral bone allografts were obtained from the bone bank after 1, 3 and 5 years of –80°C cryopreservation. Samples of each humeral head, i.e., cartilage, subchondral bone and spongy bone were histologically examined for inter- and intra-cellular changes. A three-point mechanical bending test was used on identical pieces of cortical bone to compare fresh and cryopreserved materials. Fresh-retrieved cortical bone using identically-sized segments, served as a control. Cultures were taken from each respective sample to determine contamination or sterility.Results of both the histological and mechanical testing showed that there were no significant, qualitative histological, or quantitative mechanical differences among the samples. All the cultures were negative. Therefore, based on this study's parameters, bone allografts can safely be used after a cryopreservation period of over 5 years and should not be discarded.     

The differentiation of bone marrow cells to functional T lymphocytes following implantation of thymus grafts and thymic stroma in nude and AT×BM mice

Cardiac allografts were used to compare the immunologic capacity of nude mice and adult, thymectomized, lethally irradiated, bone marrow-reconstituted (AT × BM) mice. Neither nude nor AT × BM mice were able to reject cardiac allografts of any party. However, both rejected grafts of any party following implantation of neonatal thymus or thymus from 3-week-old syngeneic mice. Irradiated syngeneic thymus grafts (800 R) were equally effective in restoring host responsiveness against allografts. In contrast, allogeneic thymus grafts restored the capacity to reject second-party heart grafts only in AT × BM mice. Second-party grafts persisted indefinitely when placed on nude mice implanted with an allogeneic, unirradiated thymus graft. Third-party grafts transplanted 17 weeks after reconstitution, however, were rejected. Irradiated nude mice given normal littermate bone marrow and simultaneously grafted with second-party thymus and heart allografts also failed to reject their second-party heart grafts. The difference in ultimate capacity to respond between AT × BM and nude mice suggests that a maturational defect exists in the nude mouse enviroment which impedes development of precursor T lymphocytes.     

Flow cytometric analysis from fish samples stored at low,ultra-low and cryogenic temperatures

Flow cytometry is a valuable tool in biomedical and animal sciences. However, equipment used for such analysis presents limitations at field conditions, suggesting then preservation procedures for future analysis at laboratory conditions. In this study, freezing at low (−20 °C), ultra-low (−80 °C) and cryogenic temperatures (−196 °C, i.e. liquid nitrogen) were used as preservation procedures of fish tissue. Samples were maintained in 0.9% NaCl or lysing solution, and stored at the temperatures above for 0 (fresh control), 60, 120 and 180 days of storage. After storage, the samples were thawed and proceeded to flow cytometric analysis. Storage at low temperatures (−20 °C), both in lysing and 0.9% NaCl, exhibited poor results when analyzed after 60, 120 and 180 days, showing noisy peaks, deviation in the DNA content and absence of peaks. Ultralow (−80 °C) and cryogenic (−196 °C) temperatures, both in lysing solution and 0.9% NaCl, showed good results and high quality of histograms. Both storage procedures gave similar histograms and DNA content in comparison with control group (fresh) even after 60, 120 and 180 days of storage, exhibiting the main peak at 2C content from diploid cells and a secondary peak at 4C derived from dividing cells. In conclusion, samples may be stored for 180 days at −80 °C and −196 °C in both, 0.9% NaCl or lysing solution. As cryogenic temperatures in liquid nitrogen permits indefinite storage, this procedure should be used for long-term preservation.     

Glycerol treatment as recovery procedure for cryopreserved human skin allografts positive for bacteria and fungi

Human donor skin allografts are suitable and much used temporary biological (burn) wound dressings. They prepare the excised wound bed for final autografting and form an excellent substrate for revascularisation and for the formation of granulation tissue. Two preservation methods, glycerol preservation and cryopreservation, are commonly used by tissue banks for the long-term storage of skin grafts. The burn surgeons of the Queen Astrid Military Hospital preferentially use partly viable cryopreserved skin allografts. After mandatory 14-day bacterial and mycological culture, however, approximately 15% of the cryopreserved skin allografts cannot be released from quarantine because of positive culture. To maximize the use of our scarce and precious donor skin, we developed a glycerolisation-based recovery method for these culture positive cryopreserved allografts. The inactivation and preservation method, described in this paper, allowed for an efficient inactivation of the colonising bacteria and fungi, with the exception of spore-formers, and did not influence the structural and functional aspects of the skin allografts.     

Banking of cryopreserved arterial allografts in Europe: 20 years of operation in the European Homograft Bank (EHB) in Brussels

Vascular allografts have been used for many years in patients with infection complications and when the patient lacks own autologous venous material. Cryopreservation has permitted the long term storage of these allografts, offering the optimal solution for particular clinical situations. For more than 20 years the European Homograft Bank has prepared, stored in the liquid nitrogen vapour below ?130 °C and distributed various types of the quality controlled arterial allografts throughout the European centers and elsewhere. The tissues are prepared according to the existing European, Belgian, Swiss and other EU countries’ regulations and standards. This paper gives an overview of this activity since 1991. During this period 1,428 batches of arteries were received from recovery centres within European Union and Switzerland and 3,941 arterial segments were evaluated. 1,250 (32 %) were discarded for morphological findings (58 %), bacteriology (31 %) and other reasons, while 2,685 or 68 % (ascending and descending aorta, arch, aortic bifurcation, iliac and femoral arteries and the non-valved pulmonary bifurcations) were cryopreserved and stored. 2,506 arteries were implanted in 1,600 patients in vascular and cardiac centers in European Union and elsewhere. The most important indications were infections (65 %), critical limb ischemia (15 %) and congenital cardiac malformations (15 %). Some allografts were used for the repair of arterial injury (2 %) or prosthetic graft thrombosis (1.5 %). 10 aortic allografts (0.4 %) were used for tracheal replacement in case of cancer. In 52 cases EHB did not fulfill the surgeon’s requests due to shortage of arterial allografts. Collaboration with vascular surgeons in the tissue recovery might improve the number, diversity and quality of vascular allografts. A multicentric study is necessary to evaluate the long-term outcome of these allografts.     

Long-term storage in liquid nitrogen does not affect cell viability in cardiac valve allografts

Liquid nitrogen is the most common medium used by tissue banks for the storage of cryopreserved heart valves. This study evaluates the effect of the length of storage on human cryopreserved heart valves. Human tissues (14 aortic and 13 pulmonary) were frozen in a controlled-rate freezer (1 °C/min) and stored in the liquid phase of a nitrogen tank for 9.1 ± 1.6 years. The preservative solution was medium M199 containing 5% human serum albumin and 10% Me₂SO. After thawing in a water bath at 42 °C, the cryoprotectant was removed. Then, fragments from vascular wall and leaflet were dissected. Explant cultures and histological studies were performed in order to assess cell viability and structural integrity. CD90 and CD31 expression was analysed in cultured cells using flow cytometry. Light microscopy, immunofluorescence staining and laser scanning confocal microscopy were used to evaluate cell viability and extracellular matrix components. Electron microscopy was used for ultrastructural study. Cell cultures could be obtained from all the specimens assayed. Cells grew from explants showing a fibroblastic phenotype. CD90 expression was common in cultured cells but a low percentage of cells expressed CD31. Histological results showed a good preservation estructure in both leaflets and vascular walls. Morphological features of cellular irreversible damage were very rare. No differences which could be due to length of allograft storage period were observed. We concluded that allografts stored in liquid nitrogen up to 13 years did not significantly undergo loss of cell viability other than that due to disinfection, freezing and thawing protocols.     

接种丛植菌根真菌对湿生植物氮磷吸收能力的影响

丛枝菌根(Arbuscular mycorrhizal,AM)真菌是一类能够与大多数陆生植物共生,并改善植物生长和氮磷吸收的微生物.湿生植物在湿地污染净化过程中起着决定性的作用,但利用AM真菌改良湿生植物氮磷吸收能力的研究鲜有报道.本研究选取了3种湿生植物千屈菜、旱伞草和黄花鸢尾,在盆栽培养的基础上,分别接种根内球囊霉(RE)、摩西球囊霉(GM)、幼套球囊霉(CE)三种AM真菌,并和空白对照比较接种AM真菌对不同植物地上、地下及整株的生物量和氮磷吸收的影响.结果表明,接种AM真菌对植物生长和氮磷吸收的影响呈现出植物间和植物内的差异,促进与抑制效应表现不一.综合AM真菌对植物生物量和氮磷吸收的促进效应,最佳AM真菌-植物的组合为:千屈菜-RE,旱伞草-RE,黄花鸢尾接种-GM.三种植物接种最佳的AM真菌后植物生物量、TN量和TP量分别提高了17.7%~29.8%、15.7%~39.0%和22.3%~62.6%.本研究为今后强化湿生植物的氮磷吸收能力提供了一种新的可选择的途径.     

Platelet concentrate as an additive to bone allografts: a laboratory study using an uniaxial compression test

Chemical cleaning procedures of allografts are destroying viable bone cells and denaturing osteoconductive and osteoinductive proteins present in the graft. The aim of the study was to investigate the mechanical differences of chemical cleaned allografts by adding blood, clotted blood; platelet concentrate and platelet gel using a uniaxial compression test. The allografts were chemically cleaned, dried and standardized according to their grain size distribution. Uniaxial compression test was carried out for the four groups before and after compacting the allografts. No statistically significant difference was found between native allografts, allografts mixed with blood, clotted blood, platelet concentrate and platelet concentrate gel regarding their yield limit after compaction. The authors recommend to chemical clean allografts for large defects, optimize their grain size distribution and add platelet concentrate or platelet rich plasma for enhancing as well primary stability as well bone ingrowth.