Stromal cell derived factor-1α enhances bone formation based on in situ recruitment: a histologic and histometric study in rabbit calvaria

Histological methods were used to assess whether in situ recruitment using stromal cell derived factor-1α (SDF-1α) enhances bone formation. Four defects were created in the calvarias of 16 rabbits and filled with: (1) a blood clot only (group C); (2) autogenous bone particles (AB, 0.2 ml) (group AB); (3) AB (0.1 ml) + bone marrow derived stromal stem cells (group ABC); or (4) AB (0.1 ml) + SDF-1α (group ABS). Bone formation was significantly greater in groups AB and ABC compared with group ABS after 2 weeks (P < 0.05). Bone formation was similar between groups AB, ABC, and ABS after 4 weeks (P > 0.05). SDF-1α is a promising candidate for in situ recruitment in bone regeneration.     

A biomechanical comparison of vascularized and conventional autogenous bone grafts

Vascularized and conventional autogenous rib grafts were used to reconstruct 6-cm ulnar defects in the forelegs of the nine dogs. Each dog served as its own control. Biomechanical torsional testing of the grafted ulnas showed that vascularized grafts were 234 percent stronger than the conventional grafts. Bone toughness (energy absorbed) was 483 percent greater in the vascularized grafts, and elastic modulus and proportional limits were 263 and 246 percent greater, respectively. We conclude that vascularized bone grafts are significantly stronger than conventional autogenous bone grafts after 3 months of healing in the dog ulna model.     

Scanning electron microscopic and light microscopic observations on morphological changes of freeze-dried bone implantation in rats: comparison with fresh autogenous bone transplantation.

Bone remodelling after the implantation of freeze-dried autogenous bone in rat parietal bone was compared with fresh autogenous bone transplantation, using a scanning electron and light microscope revealed the time intervals after transplantation/implantation. The light microscope revealed the time delay of the bone remodelling in the implantation, compared with the transplantations. The scanning electron microscope showed that the differences between the two groups were in the states of bone union and bone resorption. In the fresh bone group, the newly-formed bone filled the spaces between host and the transplanted bones at 2 to 3 weeks after the transplantation: the newly-formed bone fused and melted into the transplanted bone. New bone formation was more dominant on the bone surface in the dura mater side than in the skin side. The union was almost completed at 5 weeks. In freeze-dried bone implantation, the bone union in the contact space was very poor and the implanted bone was mainly covered by the new bone, which developed from the host bone surface in the dura mater side at 2 to 3 weeks after the implantation. What is noteworthy is that bone resorbed areas showing numerous Howship's lacunae were mainly observed on the host bone surface in the vicinity of newly-formed bone. However in freeze-dried bone implantation, the bone resorption was greater on the host and implanted bone surface than that of fresh bone transplantation: the resorption of host bone was considerably larger at certain periods after freeze-dried bone implantation. The present results show that the healing process of freeze-dried bone implantation, even though autogenous bone was used, differed from that of fresh autogenous bone transplantation, and the differences are concerned not only with time sequences but also with qualitative changes. This suggests that the host would have some different responses to the freeze-dried autogenous bone from fresh materials.     

Fresh frozen homologous bone in oral surgery: case reports

Intraoral bone defects may be treated using autologous grafts, homologous grafts, heterologous grafts or synthetic products. Autologous bone is now considered the gold standard for bone grafting procedures. Homologous fresh frozen bone, provided by bone banks, is frequently utilized by orthopaedic surgeons because it is considered a safe material from immunological and viral points of view.In the cases reported here, homologous bone was used to repair some osseous defects without changing the surgical protocol utilized for autologous bone procedures. The main advantages of this strategy are low morbidity, shorter surgical times, more comfort and less risk of infection for the patient as well as the greater availability of bone.     

Fresh cortical autograft versus fresh cortical allograft effects on experimental bone healing in rabbits: radiological, Histopathological and Biomechanical evaluation

Bone grafting is used to enhance healing in osteotomies, arthrodesis, and multifragmentary fractures and to replace bony loss resulting from neoplasia or cysts. They are source of osteoprogenitor cells and induce bone formation and provide mechanical support for vascular and bone ingrowth. Autografts are used commonly but quantity of harvested bone is limit. This study was designed to evaluate fresh cortical autograft and allograft effects on bone healing process. Twenty male White New Zealand rabbits were used in this study. In autograft group the defect was filled by fresh autogenous cortical graft, in allograft group the defect was filled by a segment of fresh allogenous cortical bone which was harvested at the time of surgery during the creation of radius bone defect. Then all surface soft tissue, such as muscle attachments, were removed from the harvested bone and changed between rabbits as a fresh allogenous cortical bone graft and was fixed by cercelage wire. Radiological, histopathological and biomechanical evaluations were performed blindly and results scored and analyzed statistically. Statistical tests did not support significant differences between two groups at the 14th and 56th postoperative day radiographically (P > 0.05). There was a significant difference radiologically for the 28th and the 42nd postoperative (P < 0.05). Autograft was superior to allograft at the 28th and 42nd postoperative day in radiological evaluation (P < 0.03). Histopathological and biomechanical evaluation revealed no significant differences between two groups.     

Tissue-engineered bone formation with cryopreserved human bone marrow mesenchymal stem cells

Bone marrow mesenchymal stem cells (MSCs) have become the main cell source for bone tissue engineering. It has been reported that cryopreserved human MSCs can maintain their potential for proliferation and osteogenic differentiation in vitro. There are, however, no reports on osteogenesis with cryopreserved human MSCs in vivo. The aim of this study was to determine whether cryopreservation had an effect on the proliferation capability and osteogenic differentiation of human MSCs on scaffolds in vitro and in vivo. MSCs were isolated from human bone marrow, cultured in vitro until passage 2, and then frozen and stored at −196 °C in liquid nitrogen with 10% Me₂SO as cryoprotectant for 24 h. The cryopreserved MSCs were then thawed rapidly, seeded onto partially demineralized bone matrix (pDBM) scaffolds and cultured in osteogenic media containing 10 mM sodium β-glycerophosphate, 50 μM l-ascorbic acid, and 10 nM dexamethasone. Non-cryopreserved MSCs seeded onto the pDBM scaffolds were used as control groups. Scanning electronic microscopy (SEM) observation, DNA content assays, and measurements of alkaline phosphatase (ALP) activity and osteocalcin (OCN) content were applied, and the results showed that the proliferation potential and osteogenic differentiation of MSCs on pDBM in vitro were not affected by cryopreservation. After 2 weeks of subculture, the MSCs/pDBM composites were subcutaneously implanted into the athymic mice. The constructs were harvested at 4 and 8 weeks postimplantation, and histological examination showed tissue-engineered bone formation in the pDBM pores in both groups. Based on these results, it can be concluded that cryopreservation allows human MSCs to be available for potential therapeutic use to tissue-engineer bone.     

Effectiveness of fresh frozen and cryopreserved homologue iliac crest grafts used in sinus lifting: a comparative study

In the last decade, several investigators have reported that autologous and homologous fresh frozen bones (FFB) are effective materials to restore alveolar ridges previous to insert dental implants. Recently we have used cryopreserved homologue grafts (CFFB). Here we reported a retrospective comparative study between implants inserted in FFB and CFFB evaluate their clinical outcome. Patients were treated with a split mouth scheme for bone grafting with FFB and CFFB and spiral family implants (SPI) were inserted in the same surgical time. Several variables (patient, grafts, anatomic site, implant, prosthetic restoration) were investigated. Implant’ failure and peri-implant bone resorption were considered as predictor of clinical outcome. A total of 84 SFIs were inserted in 12 patients. Implants were inserted to replace 8 incisors, 4 cuspids, 31 premolars and 41 molars. The mean follow-up was 14 months. Three out of 84 implants was lost (i.e. survival rate SVR = 96.4%) and no differences were detected among the studied variables. Similar result was obtained by analyzing the crestal bone resorption around implant’ neck (i.e. success rate). FFB and CFFB have high and comparable survival and success rate. Implants inserted with one step surgical procedure in native (i.e. not grafted) bone, FFB and CFFB have similar clinical outcome.     

Collection,processing and freezing of equine bone marrow cells

There is no consensus on aspects of equine bone marrow collection and processing. The study aimed to describe the collection of large volumes of bone marrow from horses of advanced age, with emphasis on bone marrow mononuclear cells (BMMCs) recovery and viability after cryopreservation. Fourteen horses, aged 3–24 years, were divided into three experiments. E1 studied the feasibility of collecting 200 mL from the sternums of horses of advanced age; E2 examined the number of cells obtained from the first and last syringe of each puncture; and E3 investigated the influence of heparin concentration on the prevention of cell aggregation, and cell viability after freezing in liquid nitrogen. Bone marrow aspirations were done with syringes pre-filled with Iscove's modified Dulbecco's medium and different concentrations of sodium heparin. BMMCs were counted, cell viability was determined, and samples were frozen. Bone marrow collection from the sternum is safe, even at large volumes and from horses of advanced age, and the number of cells recovered decreases with successive aspirations (p < 0.0001). Heparin concentration influenced cell aggregation, and recovered cells continued to be commercially viable after 150 days in frozen storage.     

Cultivation of frozen mouse bone marrow cells in agar.

The authors attempted to cultivate frozen mouse bone marrow cells in a semisolid medium. They demonstrated that the stem haematopoietic cells of frozen mouse bone marrow were capable of proliferation and of colony formation on agar. The much smaller number of colonies from frozen mouse bone marrow (about 80% fewer) compared with fresh marrow is evidence that part of the stem haematopoietic cell population retains proliferative capacity even after freezing.     

Cultivation of fresh and frozen mouse bone marrow. - Application of methyl cellulose.

The culture of cells of both fresh and frozen mouse bone marrow on methyl cellulose (MC) was approached. We used 1.5%-concentration of MC and proved the stem cells of fresh and frozen mouse bone marrow to proliferate and form haemopoietic colonies on MC. We established that the freezing process did not significantly decrease the proliferative capacity of CFU-C stem cells.     

Effect of isolation of periosteum and dura on the healing of rabbit calvarial inlay bone grafts

Little is understood about the role of the recipient site in the revascularization and incorporation of autogenous inlay bone grafts in the craniofacial skeleton. Clinical experience demonstrates that secondary complex cranial vault reconstruction performed with scarred avascular dura or poor soft-tissue coverage may undergo significant resorption, thus compromising the aesthetic outcome. This study was designed to determine the effect of isolating autogenous orthotopic inlay calvarial bone grafts from the surrounding dura and/or periosteum on graft revascularization, healing, and volume maintenance in the adult rabbit. Adult rabbits were randomized into four groups (n = 10 per group); in each rabbit, the authors created a circular, 15-mm in diameter, full-thickness cranial defect followed by reconstruction with an autogenous calvarial bone graft, which was replaced orthotopically and held with microplate fixation. Silicone sheeting (0.5 mm thickness) was used to isolate the dura (group II), the periosteum (group II), or both dura and periosteum (group IV) from the graft interface. No silicone was placed in group I. Animals were killed 10 weeks postoperatively, and calvaria were harvested to assess graft surface area, morphology, quantitative histology, fluorochrome staining, and revascularization. Grafts isolated from both the dura and periosteum exhibited significant decreases in total bone (cortical and trabecular) surface area, blood vessel count, and interface healing compared with nonisolated control grafts. Isolation of either the dura or periosteum significantly (p < 0.05) decreased blood vessel count but had no significant effect on interface healing. Isolation of the dura alone was associated with a significant (p < 0.05) decrease in graft cross-sectional surface area and dural cortical thickness compared with nonisolated control grafts, but this effect was not observed when the periosteum alone was isolated. Quantitative histology performed 10 weeks after surgery indicated that graft isolation was associated with increased marrow fibrosis and necrosis compared with nonisolated controls; it also demonstrated evidence of increased activity in bone remodeling (osteoblast and osteocyte count, new trabecular bone, and surface resorption). Triple fluorochrome staining suggested increased bone turnover in the nonisolated grafts compared with isolated grafts at 1 and 5 weeks postoperatively. This study demonstrates that isolating a rabbit calvarial inlay autogenous bone graft from the dura and/or periosteum results in significantly (p < 0.05) decreased revascularization, interface healing, and cross-sectional areas of amount of mature bone compared with nonisolated control grafts 10 weeks after surgery. At this time point, histologic examination demonstrates a paradoxical increase in bone remodeling in isolated bone grafts compared with controls. It is possible that the inhibition of revascularization results in a delayed onset of the remodeling phase of graft incorporation. However, in the model studied, it is not known whether the quantitative histologic and morphometric parameters measured in these isolated grafts exhibit a "catch-up" phenomenon at time points beyond 10 weeks after surgery. The results of this study emphasize the importance of a healthy recipient site in the healing and incorporation of calvarial bone grafts but stress the need for further investigation at later time points.     

Effect of bovine fetal growth plate as a new xenograft in experimental bone defect healing: radiological,histopathological and biomechanical evaluation

Bone grafting is used to enhance healing in osteotomies, arthrodesis, and multifragmentary fractures and to replace bony loss resulting from neoplasia or cysts. They are source of osteoprogenitor cells and induce bone formation and provide mechanical support for vascular and bone ingrowth. Autografts are used commonly but quantity of retrieved bone is limit. This study was designed to evaluate autograft and new xenograft (Bovine fetal growth plate) effects on bone healing process. Twenty male White New Zealand rabbits were used in this study. In autograft group the defect was filled by fresh autogenous cortical graft, in xenograft group the defect was filled by a segment of bovine fetal growth plate and was fixed by cercelage wire. Radiological, histopathological and biomechanical evaluations were performed blindly and results scored and analyzed statistically. Statistical tests did not support significant differences between two groups at the 14th and 28th postoperative day radiographically (P > 0.05). There was a significant difference for remodeling at the 42nd postoperative radiologically (P < 0.05). Xenograft was superior to autograft at the 56th postoperative day for radiological bone formation (P < 0.03). Histopathological and biomechanical evaluation revealed no significant differences between two groups. The results of this study indicate that satisfactory healing occurred in rabbit radius defect filled with calf fetal growth plate. Complications were not identified and healing was faster than cortical autogenous grafting. It was concluded that the use of calf fetal growth plate as a new xenograft is an acceptable alternative to cortical autogenous graft and could reduce the morbidity associated with harvesting autogenous graft during surgery.     

Lyophilized allogeneic bone tissue as an antibiotic carrier

The rising number of primary joint replacements worldwide causes an increase of revision surgery of endoprostheses due bacterial infection. Revision surgery using non-cemented implants seems beneficial for the long-term outcome and the use of antibiotic-impregnated bone grafts might control the infection and give a good support for the implant. In this study we evaluated the release of antibiotics from fresh-frozen and lyophilized allogeneic bone grafts. Lyophilized bone chips and fresh frozen bone chips were mixed with gentamicin sulphate, gentamicin palmitate, vancomycin, calcium carbonate/calcium sulphate impregnated with gentamicin sulphate, and calcium carbonate/calcium sulphate bone substitute material impregnated with vancomycin. The efficacy of each preparation was measured by drug release tests and bacterial susceptibility using B. subtilis, S. aureus and methicillin-resistant Staphylococcus aureus. The release of gentamicin from lyophilized bone was similar to the release rate from fresh frozen bone during all the experimental time. That fact might be related to the similar porosity and microstructure of the bone chips. The release of gentamicin from lyophilized and fresh frozen bone was high in the first and second day, decreasing and keeping a low rate until the end of the second week. Depending on the surgical strategy either polymethylmethacrylate or allogeneic bone are able to deliver sufficient concentrations of gentamicin to achieve bacterial inhibition within two weeks after surgery. In case of uncemented revision of joint replacements allogeneic bone is able to deliver therapeutic doses of gentamicin and peak levels immediately after implantation during a fortnight. The use of lyophilized and fresh frozen bone allografts as antibiotic carriers is recommended for prophylaxis of bone infection.     

Capacitation status of fresh and frozen-thawed buffalo spermatozoa in relation to cholesterol level, membrane fluidity and intracellular calcium

The present study was conducted to assess the capacitation status of fresh and frozen-thawed buffalo spermatozoa and its relationship with sperm cholesterol level, membrane fluidity and intracellular calcium. Semen from seven buffalo bulls (eight ejaculates each) was divided into two parts. Part I was used as fresh semen and part II was extended in Tris–egg yolk extender, equilibrated (4 °C for 4 h) and frozen at −196 °C in LN₂. The fresh and frozen-thawed spermatozoa were assessed for capacitation status using chlortetracycline (CTC) fluorescent assay, membrane fluidity using merocyanine 540/Yo-Pro-1 assay and intracellular calcium using Fluo-3 AM with flowcytometry. Results revealed a significant (P < 0.01) increase in capacitated sperm population in frozen-thawed semen compared to fresh semen (42.21% vs 14.32%). Similarly, a significantly (P < 0.01) higher proportion of frozen-thawed live spermatozoa showed high membrane fluidity (53.62% vs 25.67%) and high intracellular calcium (43.68% vs 11.72%) compared to fresh semen. The sperm cholesterol was significantly (P < 0.01) reduced after freezing–thawing as compared to fresh semen. The proportion of capacitated spermatozoa (CTC pattern B) was positively correlated with the proportion of sperm with high intracellular calcium (r = 0.81) and high membrane fluidity (r = 0.65), and negatively correlated with cholesterol level (r = −0.56) in frozen-thawed semen. The membrane fluidity was also strongly associated with the cholesterol level and intracellular calcium. The study concluded that changes in buffalo spermatozoa and established the relationship among capacitation status, sperm cholesterol level, membrane fluidity and intracellular calcium concentration in frozen-thawed spermatozoa.     

Long term follow-up of reconstruction with allogeneic mandibular bone crib packed with autogenous particulate cancellous bone marrow

Allogeneic mandibular bone processed by the deep frozen method was used as a biologic crib for mandibular reconstruction. The allogeneic mandible is biocompatible, bioresorbable, of low antigenicity and provides the morphology for symphysis contour, angle of mandible and dental arch form. The particulate cancellous bone marrow (PCBM) contains marked osteogenic potential of hematopoietic marrow, which promotes osteogenesis. The cancellous marrow graft lacks structure and requires a crib to house it during the bone regeneration to the consolidation phase. Fresh frozen mandible was hollowed out for packing with PCBM prior to securing it to the defect by a rigid fixation method. Four cases of large mandibular defects resulting from treatment of benign odontogenic tumors were reconstructed utilizing this technique. All cases showed excellent facial contour and function satisfactorily in mastication and pronunciation. Complete graft incorporation and restoration of the osseous continuities were observed for four to 12 years after the operation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Viability of maxillary bone harvesting by using different osteotomy techniques. A pilot study

The use of autogenous grafts is still considered in bone regeneration surgeries. However, the bone cell viability of such grafts after being harvested from donor sites remains a matter of debate. The aim of the present study is to evaluate particulated and block bone cell viability, in terms of presence or absence of apoptosis and necrosis, obtained from different maxillary intra-oral harvesting methods: bone scraper, rotary carbide burs and piezoelectric device. Five healthy patients were enrolled in the study. The patients required sinus augmentation by lateral window approach. The bone was harvested by the bone scraper, piezoelectric device and rotary surgical instrument. The samples were processed with the Annexin V/FITC (fluorescein isothiocyanate stain) kit and were analyzed by means of Fluoresence-Activated Cell Sorted (FACS) technique. Within the limitations of this pilot study, the results indicated that autogenous bone chips collected from the three harvesting methods presented a large percentage of apoptotic cells, although large scale production of necrotic cells was not detected. In summary, although rotary surgical instrument and piezoelectric devices are frequently used instruments for oral osteotomy, fresh autogenous bone chips collected from them did not present a viable bone cell source.     

Iliac crest fresh frozen homografts used in pre-prosthetic surgery: a retrospective study

In the case of severe jaw atrophy several options are available to restore the alveolar crest. Aim of the present study was to evaluate the resorption over time of homologous fresh frozen bone used to restore the alveolar ridge. Specifically factors influencing (1) graft survival, (2) type, and (3) degree of bone resorption were evaluated. One hundred and thirteen maxillae and 27 mandibles were grafted. The surgical techniques used were 102 inlay, 27 onlay, and 11 veneer. Measurements were taken on pre-operative, post-operative, and follow-up radiographs. Data were processed by using three statistical methods: Kaplan–Meier algorithm, Cox regression, and curve estimation. As regards graft survival, Cox regression output showed a statistically significant effect only on surgical technique (P = 0.0312) and Kaplan–Meier algorithm demonstrated a worse outcome for veneer surgical technique (Log rank test = 0.0242). The Curve estimation demonstrated an inverse correlation between degree of bone resorption over time, with a progressive decrease. In conclusion FFB is a reliable material for alveolar bone restoration with a predicable average of resorption.     

The effects of prolonged deep freezing on the biomechanical properties of osteochondral allografts

Musculo-skeletal allografts sterilized and deep frozen are among the most common human tissue to be preserved and utilized in modern medicine. The effects of a long deep freezing period on cortical bone has already been evaluated and found to be insignificant. However, there are no reports about the influences of a protracted deep freezing period on osteochondral allografts. One hundred osteochondral cylinders were taken from a fresh specimen and humeral heads of 1 year, 2 years, 3 years and 4 year old bones. Twenty chips from each period, with a minimum of 3 chips per humeral head. Each was mechanically tested by 3 point compression. The fresh osteochondral allografts were significantly mechanically better than the deep frozen osteochondral allografts. There was no statistical significant time dependent difference between the deep frozen groups in relation to the freezing period. Therefore, we conclude that, from the mechanical point of view deep freezing of osteochondral allografts over a period of 4 years, is safe without further deterioration of the biomechanical properties of the osteochondral allografts.     

Very long-term radiographic and bone scan results of frozen autograft and allograft bone grafting in 17 patients (20 grafts) a 30- to 35-year follow-up

In the early 1950s, 48 patients received bone implants from a bone bank in Tel-Hashomer Hospital that stored frozen autograft and allograft bones at temperatures less than -17 degrees C. Seventeen (35%) of these patients (20 implants), 10 men and 7 women, with a mean age of 52.4 (34-69) years were available for follow-up after a mean period of 32.5 (30-35) years. They underwent clinical examination, radiographs and bone scans to evaluate their surgical results. Fracture healing, non-union, graft resorption, osteoporosis and bone sclerosis were used as radiographic criteria for bone incorporation, and normal, increased and decreased uptake served to assess the bone scan. Based on the above criteria, the results were satisfactory in 17 (85%) and poor in 3 (15%). The three failures were after shelf operation for hip dysplasia that used two allografts and one autograft. Allogenous or a combination of allogenous with autogenous frozen bone grafts proved to be a satisfactory and durable method for filling bone defects.     

The process of bone regeneration from devitalization to revitalization after pedicle freezing with immunohistochemical and histological examination in rabbits

The pedicle freezing procedure by liquid nitrogen is a method for the reconstruction of tumor-bearing bone after malignant tumor resection. However, the regenerative mechanism of bone after the pedicle freezing procedure is unclear. We investigated the complete process from devitalization to revitalization of bone after the pedicle freezing procedure in 13 rabbits. After osteotomy the 5 mm distal femurs were immersed in liquid nitrogen, and the specimens were divided into frozen area and sub-frozen area. The bilateral femurs were harvested for evaluation of bone regeneration by histological and immunohistochemical examination (VEGF, CD31, BMP-2 and Runx2) from 1 week to 52 weeks. The diameter of operating femurs was compared with contralateral femurs from 6 weeks to 52 weeks.No viable cells could be found from 1 to 8 weeks in the frozen area, and a mean 1.83 cm necrotic range were detected in the sub-frozen area. The periosteal reaction, massive fibrous tissue and immature bone matrix invaded from the normal area to the necrotic area from 12 weeks. Subsequently, the necrotic bone was gradually replaced by newly formed bone by creeping substitution, with endochondral and intramembrane bone formation. The diameter of frozen femurs was significantly larger than the contralateral femur at the same period from 8 weeks to 52 weeks (P < 0.01). All immunohistochemical factors were positively expressed in both areas at different time points. The active osteoblasts and microvessel migrated from marrow cavity and periosteum into dead bone. This study suggested that the frozen bone not only provides a scaffold but also possesses excellent osteoinductive properties.