Evaluation of autogenous bone grafts, particulate or collected during osteotomy with implant burs: histologic and histomorphometric analysis in rabbits

PURPOSE: The aim of this study was to evaluate bone regeneration in bone cavities filled with particulate autogenous bone either harvest in blocks and subjected to milling procedures or collected during osteotomy with implant burs. MATERIALS AND METHODS: In 12 rabbits, 3 noncritical unicortical cavities 7 mm in diameter were prepared with a trephine drill on the right tibia. The cavities were filled respectively with particulate autogenous bone achieved with a manual bone crusher (particulate group), with particulate autogenous bone obtained using bone collector during osteotomy (collected group), and with blood clot (control group). Animals were sacrificed at 7, 15, and 30 days after surgery (4 animals for each time period). The sections were examined by histologic and histomorphometric analysis. RESULTS: At 7 days, the samples were filled by coagulum, and bone particles were observed only in the collected (24%) and particulate groups (44.75%). At 15 days, there was connective differentiation in all groups, with presence of grafted bone particles and onset of newly formed bone in the collected (38.88%) and particulate groups (46.0%). At 30 days, there was bone fill (immature trabecular bone) of the cavities in the control (50%), collected (64.63%) and particulate groups (66%). CONCLUSION: No significant difference was demonstrated between noncritical unicortical bone defects in rabbit tibiae filled with particulate bone harvested as a block and subjected to milling and those filled with bone collected during osteotomy with implant drills when the defects were observed up to 30 days following their creation.     

Repair of cranial bone defects with calcium phosphate ceramic implant or autogenous bone graft

Autogenous bone grafts have frequently been used in the treatment of bone defects; however, this procedure can cause clinical complications after surgery. Besides, the amount of available bone is sometimes insufficient. Therefore, synthetic biomaterials have been researched as an alternative to autogenous bone graft implants. The objective of this study was to evaluate the repair of bone defects treated with compact autogenous bone graft or porous calcium phosphate ceramics. Three defects 3 mm in diameter were produced in the skull of 21 rats. One the defects was produced in the frontal bone, which remained empty, while the others were produced in the right and left parietal bones, which were filled respectively with ceramics and autogenous bone graft. The animals were sacrificed 1, 2, 4, and 24 weeks after surgery and analyzed by light microscopy and radiography. In the twenty-fourth week, the defects filled with autogenous bone graft and ceramics had similar volumes of newly formed bone tissue. The ceramics offered favorable conditions to bone tissue growth. Thus, we concluded that the calcium phosphate ceramic implant proved to be effective in repairing defects produced in the skull of rats.     

Use of BioGran and Calcitite in bone defects: histologic study in monkeys (Cebus apella)

The present study compares the biologic behavior of BioGran and Calcitite as fillers for surgical cavities in the mandibles of 4 adult monkeys (Cebus apella). The surgical cavities were prepared through both mandibular cortices, with a diameter of 5 mm, in the angle region. Two cavities were prepared on the right side and 1 on the left and divided into 3 groups: R1 sites were filled with bioglass (BioGran), R2 sites were not filled, and L sites were filled with hydroxyapatite (Calcitite). After 180 days the animals were sacrificed and the specimens were removed for histologic processing. Results showed no bone formation in group R2 (empty cavities). BioGran-treated sites showed bone formation and total repair of the bone defect, and the bioglass particles were almost totally resorbed and substituted by bone. The few remaining crystals were in intimate contact with newly formed bone. Calcitite did not allow bone formation, and granules inside the cavities were involved by connective tissue. Based upon those results, the authors concluded that bioglass resulted in total obliteration of the surgical cavity with bone and hydroxyapatite was present in a large amount and involved by connective tissue, without bone formation.     

Comparative analyses among interfaces of some ceramic materials and bone in sheep

Aim of this work is the evaluation of "in situ" implants in an animal model to study the interfaces that some ceramic materials for dental bone defects develop with bone and to check which material is more osteoconductive. In a sheep's jaw, eight holes were drilled and filled with six ceramic materials in granular shape. Two bilateral holes were left empty as reference. The ceramic materials were: porous tricalcium phosphate (TCP), porous hydroxylapatite (HA) and four bioactive glasses. The glasses differ for doping agents that affect the velocity of biodegradation in the living body. Monthly radiographs were taken and the X-ray pictures analyzed by means of a Video Display Computer in order to quantify the optical density changes occurred in the holes. After 4 months implantation, the segments of the jaw containing the materials were fixed in paraformaldehyde, embedded in methylmethacrylate and sectioned. The results obtained under the microradiograph, the SEM and the X-ray microprobe showed a good bone repair only with TCP granules. A great degradation was seen in HA granules and particularly in glasses. The degradation modified the structure and the composition of the glass granules, but it was not followed by a consequent bone deposition.     

Histological and histomorphometric analyses of calcium phosphate cement in rabbit calvaria

PURPOSE: To assess bone regeneration in critical sized defects in the rabbit calvarium, filled with the bone substitute calcium phosphate cement. MATERIAL AND METHODS: Circular bone defects (8mm) were made in both parietal bones of 10 rabbits. One of the defects was filled with the calcium phosphate cement, and the other received autogenous bone harvested from the calvaria. The animals were killed at 3 or 6 weeks (n=5). Data analysis included qualitative assessment of the calvarial specimens and histomorphometric analysis was used to quantify the amount of new bone within the defects. RESULTS: The microscopic analysis of the samples showed bone healing with both calcium phosphate cement and autogenous bone graft. Data obtained from the histomorphometric analysis were statistically analyzed using 2-way analysis of variance and the Tukey's test. Data analysis showed that the autogenous bone graft had significantly more new bone compared with calcium phosphate cement at 3 and 6 weeks. Calcium phosphate cement at 6 weeks presented similar results to autogenous bone at 3 weeks. Both treatments presented an increase in bone healing with time. CONCLUSION: Treatments allowed bone regeneration that increased with time, however surgical cavities treated with the autogenous graft had more bone formation than those with calcium phosphate cement.     

Bioactive glass particulate material as a filler for bone lesions

Calcium-phosphate ceramic particulates are often used as filler material for enhanced repair of dental bone defects. Although evidence of bone ingrowth in the scaffold of these particles has been described, it is not observed consistently. Fibrous tissue often encapsulates these particles, which can subsequently become dispersed into the surrounding tissues or even exfoliated. The aim of the present study was to evaluate bioactive glass granules (Biogran) as a filler for osseous lesions, and to compare them with two commercially available Hydroxylapatite (HA) granules. The particulates were implanted in the jaws of five beagle dogs, resected and evaluated after 1, 2, 3, 6 and 12 months of implantation. Histological analysis revealed an improvement in repair of all the lesions. A massive osteoconductive bone growth was seen near the walls of the bony cavities, but in greater amounts around the bioactive glass granules than around the HA materials. On top of this massive growth a trabecular bone growth was observed in the centre of the bony cavities. These trabeculae were associated with the glass particles, which exhibited osteophilic properties, while fibrous tissue separated the bone tissue from the HA particles. The centres of many of the particles are excavated, and are subsequently filled by newly formed bone tissue. This internally formed bone tissue is not necessarily connected to the surrounding bone tissue, and functions as a nucleation site for further bone repair. For the mesenchymal cells within the eroded glass particles this inner environment acts as a stimulus to differentiate into osteoblasts and to start their osteogenetic potential. This phenomenon was not observed around the HA materials. If the latter were surrounded by fibrous tissue, disintegration of the surface by giant cells was observed.     

A comparative analysis of bone formation induced by human demineralized freeze-dried bone and enamel matrix derivative in rat calvaria critical-size bone defects

BACKGROUND: Human demineralized freeze-dried bone allograft (DFDBA) and enamel matrix derivative (EMD) have been used with varying success for the treatment of bone and periodontal defects. The purpose of this study was to compare qualitatively and quantitatively the bone formation induced by DFDBA and EMD to that of a positive control, recombinant human bone morphogenetic protein 2 (rhBMP-2), in the 8-mm rat calvaria critical-size bone defect. METHODS: Five groups of five rats each were used. The two test groups were DFDBA and EMD. A negative control consisted of a defect without any biomaterial implanted, a positive control consisted of a defect filled with collagen carrying rhBMP-2, and a non-surgical control consisted of the intact rat calvaria. Eight weeks after implantation of the biomaterials, histologic analysis was used for qualitative assessments and microcomputed tomography was used for quantitative assessments of bone formation. Statistical evaluation was performed by analysis of variance followed by the Fisher's least significant difference multiple-comparison test. RESULTS: In the negative control and EMD groups, the histologic analysis showed no bone formation within the center of the defect and limited bone repair at its margins. In the DFDBA group, granules of DFDBA were still present 8 weeks after implantation, and a limited degree of osteoinduction was seen at the center of the defect. The microcomputed tomography quantitative analysis showed a limited capacity of DFDBA and EMD to induce bone formation, and no statistically significant difference was detected among DFDBA, EMD, and the negative control. On the contrary, the positive control (rhBMP-2) consistently showed regeneration of bone throughout the critical-size defects. CONCLUSION: Unlike rhBMP-2, DFDBA and EMD had limited ability to induce bone formation in the rat calvaria critical-size bone defect; therefore, they may not be effective as bone-regenerative therapy for critical-size defects.     

Bone healing and graft resorption of autograft, anorganic bovine bone and beta-tricalcium phosphate. A histologic and histomorphometric study in the mandibles of minipigs

OBJECTIVE: The purpose was to qualitatively and quantitatively compare the bone formation and graft resorption of two different bone substitutes used in both orthopedic and oral surgery, with autogenous bone as a positive control. MATERIALS AND METHODS: Three standardized bone defects were prepared in both mandibular angles of 12 adult minipigs. The defects were grafted with either autograft, anorganic bovine bone (ABB), or synthetic beta-tricalcium phosphate (beta-TCP). Sacrifice was performed after 1, 2, 4, and 8 weeks for histologic and histomorphometric analysis. RESULTS: At 2 weeks, more new bone formation was seen in defects filled with autograft than with ABB (P approximately 0.0005) and beta-TCP (P approximately 0.002). After 4 weeks, there was no significant difference between beta-TCP and the two other materials. Defects grafted with ABB still exhibited less bone formation as compared with autograft (P approximately 0.004). At 8 weeks, more bone formation was observed in defects grafted with autograft (P approximately 0.003) and beta-TCP (P approximately 0.00004) than with ABB. No difference could be demonstrated between beta-TCP and autograft. beta-TCP resorbed almost completely over 8 weeks, whereas ABB remained stable. CONCLUSION: Both bone substitutes seemed to decelerate bone regeneration in the early healing phase as compared with autograft. All defects ultimately regenerated with newly formed bone and a developing bone marrow. The grafting materials showed complete osseous integration. Both bone substitutes may have a place in reconstructive surgery where different clinical indications require differences in biodegradability.     

Effect of adding resorbable calcium sulfate to grafting materials on early bone regeneration in osseous defects in rabbits

This experiment was designed to study the osteogenic potential of adding medical-grade resorbable calcium sulfate mixture to grafting materials for filling osseous defects. Twelve New Zealand rabbits were divided into an active group of 10 animals and a control group of 2 animals. The median condyle of each femur was drilled to create 8-mm-deep cavities. Active osseous defects consisted of 20 cavities and were filled with Osteograf, BOP, or Capset (calcium sulfate) alone; a mixture of Osteograf and Capset; or a mixture of BOP and Capset. Osteograf and BOP were each mixed with Capset in a 4:1 ratio. Each grafting material filled 4 osseous cavities, and 4 osseous cavities were left unfilled to act as controls. The observation period was 8 weeks. Block sections of the femoral heads were prepared for decalcified histologic assessment. It appeared that mixing grafting materials with calcium sulfate powder in a 4:1 ratio, respectively, facilitated the process of osteogenesis and increased new bone bonding to remnants of the grafting materials, in spite of the poor osteoconductive property of BOP and moderate osteoconductivity of Osteograf. However, calcium sulfate material alone is not recommended for use as a bone filler.     

The effect of platelet-rich plasma on early and late bone healing: an experimental study in goats

Four round critical-size defects were made in the foreheads of 20 goats. The defects were filled with autogenous particulate cancellous bone, in which 1 ml of platelet-rich plasma (PRP) was added in two of the four defects of each goat. The goats were divided into four subgroups of five goats each, which were killed after 1, 2, 6 and 12 weeks. The results of histologic and histomorphometric examination showed that early and late bone healing was not enhanced when PRP was used.     

Enhancement of bone regeneration and graft material resorption using surface-modified bioactive glass in cortical and human maxillary cystic bone defects

PURPOSE: Bioactive glass bonds to bone through a calcium phosphate layer that mimics the structure of the mineral phase of bone. Formation of this layer is inhibited in the presence of serum protein. The authors hypothesize that creation of a calcium phosphate layer on the surface of bioactive glass before implantation will enhance bone regeneration and graft material resorption in bone defects. MATERIALS AND METHODS: Bioactive glass particles covered with a layer of amorphous calcium phosphate (BG-ACP), bioactive glass particles covered with a layer of hydroxycarbonate apatite (BG-HCA), and unmodified bioactive glass particles (as a control) were prepared and implanted in cortical bone defects in dogs or in human maxillary cavities. Ungrafted sites were also used as a control. RESULTS: Histomorphometric analyses showed significantly more bone tissue regeneration and graft material resorption in the defects filled with BG-HCA than in those filled with BG-ACP or unmodified bioactive glass (P < .0001). Moreover, measurements of radiographic density of the grafted areas suggested a higher rate of bone regeneration in defects filled with the modified bioactive glass than in those filled with unmodified bioactive glass or in the ungrafted control. Bone formation was significantly greater in defects filled with unmodified bioactive glass particles than in ungrafted defects. DISCUSSION: The enhancement of bone regeneration could be explained by the ability of the apatite layer to facilitate bone adsorption and enhance calcium release, which stimulates osteoblast differentiation and bone formation. CONCLUSION: Results of both the clinical and animal studies suggest that the use of surface-modified bioactive glass covered with a hydroxycarbonate apatite layer has the potential to accelerate bone formation and graft material resorption better than unmodified bioactive glass.     

Hydroxyapatite cement (BoneSource) for repair of critical sized calvarian defects--an experimental study.

AIM: In an experimental study the hydroxyapatite cement BoneSource was tested for the ability in relation to the defect size and for its resorption properties. MATERIAL AND METHODS: In an animal study, BoneSource was applied to repair bicortical defects of different sizes in frontal bones of six Goettingen minipigs. The area was evaluated radiographically and histologically 12, 18 and 40 weeks postoperatively. RESULTS: After 40 weeks approximately 90% of the hydroxyapatite cement had been resorbed and replaced with bone. After 12 weeks, approximately 30% had been degraded, and 40% after 18 weeks. In small bone defects, the mucosa of the frontal sinus lined the bone substitute (BoneSource). In the reconstruction of large areas, a membrane was used to stabilize the material. Despite this membranous support, BoneSource material prolapsed into the frontal sinus. DISCUSSION: Hydroxyapatite cement is for the repair of bone defects. It can be moulded to shape the reconstruction. Its use is limited by the defect size and the need for a dry bed. CONCLUSION: BoneSource is well suited to repair small defects with proven high biocompatibility. However, in large defects, the material is not sufficiently stable.     

Osteoconductive Properties of β-Tricalcium Phosphate Matrix, Polylactic and Polyglycolic Acid Gel, and Calcium Phosphate Cement in Bone Defects

ABSTRACT: Extensive bone defects in maxillofacial region can be corrected with autogenous grafts; otherwise, the disadvantages of the therapeutics modality take the research for new bone substitutes. The aim of the study was to evaluate and compare the osteoconductive properties of 3 commercial available biomaterials. A total of 30 calvarial defects (5-mm diameter) were randomly divided into 5 treatment groups, with a total of 6 defects per treatment group (n = 6). The treatment groups were as follows: 500 to 1000 μm β-tricalcium phosphate (β-TCP), polylactic and polyglycolic acid (PL/PG) gel, calcium phosphate cement, untreated control, and autograft control. The evaluations were based on histomorphometric analysis at 60 postoperative days. The results have shown that β-TCP and autograft control supported bone formation at 60 postoperative days. β-Tricalcium phosphate showed the highest amount of mineralized area per total area and statistically significant compared with PL/PG, calcium phosphate cement, and untreated control groups. The PL/PG gel does not have osteoconductive properties and performed similar to empty control. Calcium phosphate cement showed higher number of multinucleated giant cells around the sites of the biomaterial and showed newly formed bone only at the edges of the biomaterial, without bone formation within the biomaterial. The findings presented herein indicate that bone formation reached a maximum level when rat calvarial defects were filled with β-TCP at 60 postoperative days. Further studies should be conducted with β-TCP to understand the potential of this biomaterial in bone regeneration.     

The use of hydroxyapatite and autogenous cancellous bone grafts to repair bone defects in rats

Bone grafts are frequently used in the treatment of bone defects. Bone harvesting can cause postoperative complications and sometimes does not provide a sufficient quantity of bone. Therefore, synthetic biomaterials have been investigated as an alternative to autogenous bone grafts. The objective of this study was to evaluate the repair of bone defects by autogenous cancellous bone grafts or porous bioceramic discs of hydroxyapatite/phosphate cement mixture. Two 5-mm diameter defects were made in the skulls of rats and filled with the bioceramic material or cancellous bone. The rats were sacrificed 2, 4, 8 and 24 weeks after surgery and tissue samples were analyzed by radiography and histology. By the 24th week, the defects filled with autogenous cancellous bone grafts or bioceramic material showed similar volumes of bone tissue within the defect. However, defects treated with bioceramic material were almost completely closed as a result of the joining of ceramic fragments and the neoformed bone tissue, while those filled with autogenous grafts showed several areas filled with connective tissue. These results indicated that the osteointegration of bioceramic fragments allowed the reconstruction of parietal bone defects without the need for a bone graft.     

Oral implants placed in bone defects treated with Bio‐Oss®, Ostim®‐Paste or PerioGlas: an experimental study in the rabbit tibiae

Objectives: To compare the histological features of bone filled with Bio‐Oss^®, Ostim‐Paste^® or PerioGlas placed in defects in the rabbit tibiae by evaluating bone tissue composition and the integration of titanium implants placed in the grafted bone. Material and methods: Two cylindrical bone defects, about 4 mm in diameter and 6 mm in depth, were created in the tibiae of 10 rabbits. The defects were filled with either Bio‐Oss^®, PerioGlas, Ostim^®‐Paste or left untreated, and covered with a collagen membrane. Six weeks later, one titanium sandblasted and acid‐etched (SLA) implant was inserted at the centre of each previously created defect. The animals were sacrificed after 6 weeks of healing. Results: Implants placed in bone previously grafted with Bio‐Oss^®, PerioGlas or Ostim^®‐Paste obtained a larger extent of osseointegration, although not statistically significant, than implants placed in non‐grafted bone. The three grafting materials seemed to perform in a similar way concerning their contribution towards implant osseointegration. All grafting materials appeared to be osteoconductive, thus leading to the formation of bridges of mineralized bone extending from the cortical plate towards the implants surface through the graft scaffold. Conclusions: Grafting with the above‐mentioned biomaterials did not add any advantage to the osseointegration of titanium SLA implants in a self‐contained defect.     

Effect of platelet-rich plasma on bone healing of autogenous bone grafts in critical-size defects

Aim: This study histologically analysed the effect of autogenous platelet-rich plasma (PRP), prepared according to a new semiautomatic system, on healing of autogenous bone (AB) grafts placed in surgically created critical-size defects (CSD) in rabbit calvaria.
Material and Methods: Sixty rabbits were divided into three groups: C, AB and AB/PRP. A CSD was created in the calvarium of each animal. In Group C (control), the defect was filled by blood clot only. In Group AB (autogenous bone graft), the defect was filled with particulate autogenous bone. In Group AB/PRP (autogenous bone graft with platelet-rich plasma), it was filled with particulate autogenous bone combined with PRP. All groups were divided into subgroups ( n =10) and euthanized at 4 or 12 weeks post-operatively. Histometric and histologic analyses were performed. Data were statistically analysed ( anova , t -test, p <0.05).
Results: Group C presented significantly less bone formation compared with Group AB and AB/PRP in both periods of analysis ( p <0.001). At 4 weeks, Group AB/PRP showed a statistically greater amount of bone formation than Group AB (64.44 ± 15.0% versus 46.88 ± 14.15%; p =0.0181). At 12 weeks, no statistically significant differences were observed between Groups AB and AB/PRP (75.0 ± 8.11% versus 77.90 ± 8.13%; p >0.05). It is notable that the amount of new bone formation in Group AB/PRP at 4 weeks was similar to that of Group AB at 12 weeks ( p >0.05).
Conclusion: Within its limitation, the present study has indicated that (i) AB and AB/PRP significantly improved bone formation and (ii) a beneficial effect of PRP was limited to an initial healing period of 4 weeks.     

Effects of platelet-rich plasma on bone healing in combination with autogenous bone and bone substitutes in critical-size defects. An animal experiment

OBJECTIVES: An animal study was carried out to investigate the influence of platelet-rich plasma (PRP) on the regeneration of bony defects. MATERIAL AND METHODS: Critical-size defects in the forehead region of a mini-pig were filled with randomly distributed combinations of autogenous bone, tricalcium-phosphate granules (CeraSorb), bovine spongious blocks (BioOss) and a bovine bone inducing collagenous sponge (Colloss) with and without PRP in two preparations (Cusasan, 3i). The animals were killed after 2, 4 and 12 weeks. The specimens were evaluated microradiographically and immunohistologically. RESULTS: Autologous bone (38 +/- 9.9%) and Colloss (52.6 +/- 4.0%) showed the highest remineralization rates at 2 weeks. The initial high expression of BMP-2 in the Colloss-group gives evidence of an early initiation of bony regeneration. At 2 weeks PRP ad modum 3i was able to enhance bone healing significantly (P=0.028) only when applied in combination with autogenous bone (62.8 +/- 1.6%). Four weeks after surgery, both PRP preparations did no longer increase bony regeneration in the autogenous groups. The osteoconductive effect of Bio-Oss (38.7 +/- 5.5%) and CeraSorb (41+/-4.9%) was remarkable as well 4 weeks after surgery. Nevertheless, the addition of PRP hardly influenced bony regeneration, ceramic degradation or cytokine expression when bone substitutes were applied. At 12 weeks, the level of reossification had adjusted similarly in all groups. CONCLUSION: PRP did not add additional benefit when xenogenic bone substitutes were used. However, a significant effect on bone regeneration was found in the autogenous group initially when PRP is added.     

Three-dimensional printing of bone repair and replacement materials: impact on craniofacial surgery

Solid freeform fabrication techniques such as direct write technology can be used to fabricate tissue-engineering scaffolds in 3 dimensions with high levels of reproducibility and precision. These can comprise complex structures made of osteoconductive, remodelable lattices to conduct bone ingrowth and solid barriers to prevent soft tissue invasion. As such, they act as a combination of bone graft and barrier membrane. Results from animal studies have shown that these structures fill rapidly with healing bone and can conduct bone across critical-size defects to fill large defects in rabbit skull. Results indicate that this technology can be used to produce both off-the-shelf and custom-fabricated bone graft substitutes. These may initially be used to restore alveolar ridge defects, but could also be used, in the future, to repair or replace complex craniofacial bone defects such as cleft palate defects. In the more distant future, these technologies could be combined with controlled-release bioactive substances such as growth factors and pharmaceuticals to regenerate complex structures comprising multiple tissue types.     

Bone defect regeneration with bioactive glass implantation in rats

The main goal of this study was to histologically evaluate the healing of surgically created defects on the tibiae of adult male rats after implantation of two types of bioactive glass. Sixteen adult Wistar rats (body weight of 300g) were divided into two groups: PerioGlas (PG) (n=8) and BioGran (BG) (n=8). Unicortical bone defects with 3-mm diameter were performed in both tibiae of the animals and filled with two types of glass particles. The rats were then sacrificed at 7, 14, 30 and 60 days, and the tissues were prepared for histological processing, sectioning, and staining with hematoxylin and eosin, as well as Mallory trichrome, and analyzed under light microscope. Within 7-14 days, both groups presented connective tissue septa with new bone formation, more intense in the PG group. In the subsequent periods (30 and 60 days), both groups presented more mature bone tissue around the glass particles. Bone trabeculae formed in all experimental periods were juxtaposed to the glass particles. It can be concluded that both materials promoted comparable bone formation over the entire extension of the defect, independently of the size of the granules, thus confirming their biological osteoconductive property.     

Repair of bone defects treated with autogenous bone graft and low-power laser

Because bone healing at the graft site is similar to a fracture repair, the purpose of the present study was to evaluate the effects of low-power laser irradiation on the repair of rat skull defects treated with autogenous bone graft. A defect measuring 3 mm in diameter was produced in the left parietal bone and filled with an autogenous bone graft obtained from the right parietal bone. The animals were divided into 3 groups of 20 rats each: nonirradiated control, irradiated with 5.1 J/cm, and irradiated with 10.2 J/cm. The laser (2.4 mW, 735 nm, 3.4 x 10 W/cm, 3-mm spot size) was applied three times per week for 4 weeks. Greater volume of newly formed bone was observed in the irradiated group with 10.2 J/cm. In both irradiated groups, a greater volume of newly formed bone occurred only in the first 2 weeks. The results demonstrated that laser irradiation at the grafted site stimulated osteogenesis during the initial stages of the healing process in a skull defect of the rat and that this effect was dose dependent.