Histologic Evaluation of Osseous Regeneration Following Combination Therapy With Platelet-Rich Plasma and Bio-Oss in a Rat Calvarial Critical-Size Defect Model

Inflammation and alveolar bone loss are hallmarks of periodontal disease, and historically, regenerative attempts have involved a myriad of techniques and materials usually consisting of bone grafts and/or barrier membranes to repair bony defects.¹  While great strides have been made in periodontal research, novel therapies that improve bone regeneration are still needed, and combining treatments that have already demonstrated partial bone restoration may offer greater efficacy than single therapies.

Platelet-rich plasma (PRP), which provides an enhanced concentration of platelets compared with blood, has gained wide interest as a therapy for soft and hard tissue injuries. Blood platelets are an invaluable source of growth factors that modulate critical cellular events involved in wound-healing responses.²  In addition, many of the growth factors released from activated platelets are osteoinductive.³  Examples of growth factors found in PRP that may aid in bone regeneration are platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-β), and insulin-like growth factors (IGF; IGF-I and IGF-II).⁴  An added benefit of using PRP as a potential therapy for bone loss is that PRP is autologous, nontoxic, and nonimmunoreactive.

Bone substitutes such as Bio-Oss (Ed Geistlich Sons, Wolhusen, Switzerland), a bovine bone derivative, have demonstrated efficacy in bone regeneration. Bio-Oss is a natural material made of anorganic bovine bone containing carbonate apatite with macro- and microconfigurations closely parallel to that of the structure of human bone.⁵  One advantage of using Bio-Oss is that Bio-Oss has been completely deorganified by a proprietary extraction process that makes it free of antigenicity.⁶  Bio-Oss also possesses osteoconductive properties that may aid in bone formation in periodontal bone defects.⁷  A recent study in a canine model of dental implants indicated that bone regeneration in groups that received Bio-Oss in the defect was similar to that in autologous bone.⁸  However, previous studies have indicated that Bio-Oss is not completely resorbable, and the amount of bone regeneration has been variable.⁹  A Cochrane review of the clinical trial literature for Bio-Oss and other bone augmentations therapies, alone or in combination, for dental implants suggests that many studies were not powered sufficiently to detect true benefits.¹⁰  Thus, further studies are necessary to dissect the potential benefits of Bio-Oss, particularly in combination with other materials.

Previous research has addressed the use of growth factors, alone or in combination with Bio-Oss or other grafting materials, to enhance bone regeneration. Bio-Oss may provide an appropriate carrier vehicle for growth factors to allow for rapid repair of bony defects. In an in vitro study, Tsay et al¹¹  observed that PRP, combined with different bone substitutes (including Bio-Oss) and activated with thrombin receptor activator peptide-6, retained significant growth factor levels, allowing for a longer growth factor release for up to 14 days. The effects of bone substitutes on the repair of critical-size defects (CSDs) in rat calvarial, a model for craniomandibulofacial nonunions, has been used routinely to identify potential therapies to enhance bone growth.¹²  The combination of osteoinduction by PRP and osteoconduction with Bio-Oss could potentially enhance bone regeneration and serve as a model for future studies.

The current study combined PRP with Bio-Oss to engineer a grafting combination that may promote bone regeneration to a greater extent than each material used separately in the CSD model. The major objectives of the current study were to analyze whether a combination of PRP and Bio-Oss would enhance bone regeneration and/or close the CSD more efficiently than either material alone.

Animal care was delivered in accordance with the guidelines established by, and oversight provided by, the Institutional Animal Care and Use Committee, Dwight David Eisenhower Army Medical Center, Fort Gordon, Ga. Food (Teklad Rodent Blocks, Harlan, Indianapolis, Ind) and water were available ad libitum.

Forty-eight adult male Sprague-Dawley rats (Harlan), of the same genetic strain and weighing 325–375 g, were quarantined for 7 days then randomly divided into 4 groups of 12 rats each.

The PRP was prepared in the following manner: citrated blood (0.105 M sodium citrate) was obtained from donor rats of the same experimental population. Animals were anesthetized intraperitoneally with 65 mg/kg ketamine and 7 mg/kg xylazine. Anesthetic depth was monitored to ensure that the procedure was painless. Isoflurane anesthesia via a face mask was applied when necessary. Blood was obtained via cardiac puncture or via the caudal vena cava after a ventral midline incision was made. Exsanguinated animals were then euthanized, if necessary, by placement in a CO₂ chamber. We obtained 10 to 12 mL of blood in this manner.

The protocol for PRP preparation was patterned after the protocol used by the Hematology Department at Eisenhower Army Medical Center. The blood was decanted into centrifuge tubes and centrifuged in a Beckman Model J-6M centrifuge (Beckman Coulter, Inc, Fullerton, Calif) at 150g for 10 minutes at 20°C. The PRP or buffy coat was decanted into a small sterile beaker. Approximately 1 mL of PRP was obtained. Approximately 100 μL of whole blood and PRP were collected to determine platelet counts using an Abbot Cell-Dyn 3500 hematology analyzer (Abbott Diagnostics, Santa Clara, Calif) with the parameters set for rats. The coagulation process was initiated by adding a mixture of 1000 units of topical bovine thrombin (both from Sigma Chemical Co, St Louis, Mo) in 10% calcium chloride per milliliter of PRP. This mixture was added drop-wise until the PRP formed a gel, and then it was added to the surgical defect (as described later).

Bio-Oss, particle size 0.25–1.0 mm, was reconstituted in 1 mL of saline for use in this study. To create the PRP plus Bio-Oss implant material, the Bio-Oss was hydrated with excess PRP. The calcium chloride and bovine thrombin mixture was then added to form a gel with internalized Bio-Oss particles, and added to the defect.

Cranial CSD was created as previously described.^13–14  Briefly, animals were anesthetized intraperitoneally with a cocktail of ketamine HCl, 100 mg/mL (65 mg/kg), and xylazine HCl, 20 mg/mL (7 mg/kg), via a 25-gauge needle. Sterile eye lubricant ointment was placed in the eyes. Under sterile conditions, the skin overlying the calvarium was shaved with a #40 blade and the surgical site cleaned with Novalsan scrub (Zoetis Inc, Kalamazoo, Mich) and sterile water. An autoclaved 3-inch stockinette was rolled over the prepared rat using aseptic technique, and the head was exteriorized by cutting a hole in the stockinette and pulling the head through. The anesthetized rat was placed in a Stoelting rodent stereotaxic device (Stoeling Co, Wood Dale, Ill) to secure the head during the surgical procedure. The stereotaxic device was fitted with a specialized anesthesia nose cone, through which 0.25%–0.50% isoflurane in 1 L/min oxygen was administered and adjusted to maintain an appropriate anesthetic plane. A midline incision was made from the middle of the nasal bones to the posterior nuchal line. Full-thickness flaps with periosteum were reflected laterally. An 8-mm craniotomy was made utilizing a dental handpiece at slow speed with a sterilized 8-mm diamond grit trephine bur (Continental Diamond Tool Corp, New Haven, Ind) and copious saline irrigation. Care was taken to avoid the underlying dura. The edges of the bony defect were smoothed using a #2 round bur with saline irrigation as needed. A 9-mm diameter polytetrafluoroethylene (PTFE) membrane (Fluoropore Membrane Filters, pore size 0.5 μm, Millipore Corp, Bedford, Mass) was placed deep to the cranium and superficial to the dura with the reinforced side adjacent to the dura. The membrane was adjusted to achieve an overlap covering the defect.

After the CSD surgeries, groups were treated as follows: 1) no material was placed in the defect (surgical control group); 2) PRP (500 μL) was placed into the defect (PRP group); 3) Bio-Oss (40 mg) was placed into the defect; 4) PRP plus Bio-Oss gel (40 mg Bio-Oss, saturated with PRP, then coagulated) was placed into the defect. The saline-moistened treatment material, if any, appropriate to each study group was carefully placed into the defect. A second 10-mm diameter PTFE membrane was placed superficial to the outer cranium and deep to the periosteum so the membrane edges overlapped the defect by 1 mm. The periosteum and temporalis muscle were repositioned to cover the outer surface of the membrane and sutured with continuous 4-0 Vicryl sutures on a cutting needle. The skin was closed with interrupted sutures using 4-0 Vicryl on a cutting needle. After the surgical procedures, the animals were observed for any signs of pain or distress. If analgesics were indicated, buprenorphine (0.1–0.5 mg/kg intraperitoneally via a 25-gauge needle) was administered subcutaneously every 12 hours until signs of pain subsided.

At 8 weeks after surgery, the animals were euthanized by CO₂ asphyxiation. The cranium was removed, and placed in 10% formalin (Fischer Chemicals, Fair Lawn, NJ). The calvaria were radiographed using a Faxitron Series 43807N X-Ray System (Hewlett Packard, Wheeling, Ill) soft x-ray analysis technique (time, 15 seconds; kilovolt peak, 35; milliamps, 2.5) for radiomorphic comparisons.

The specimens were then sectioned sagitally to yield 2 samples per specimen, and 1 of these samples was demineralized for 10 days using Cal-EX Decalcifying Solution (Fischer Chemicals). Each specimen was placed in a cassette, imbedded in paraffin wax, sectioned at 3-μm thickness on a 2030 Biocut microtome (Leica, Reichert-Jung, Nussloch, Germany), and stained with hematoxylin and eosin stain. Histologic slides were scanned using a Photometrics Coolsnap Fx camera (Roper Scientific Inc, Tucson, Ariz) attached to a Nikon Diaphot 300 inverted microscope (Southern Micro Instruments, Atlanta, Ga) and SimplePCI Imaging (Compix Inc, Imaging Systems, Cranberry Township, Pa), at a ×40 power. Histology sections were then analyzed qualitatively by light microscopy at a magnification of ×40. New bone growth, as evidenced by the presence of osteoblasts, and amount of closure of the CSD were observed and documented for each histology section.

The mean whole-blood platelet count of 500 625 ± 3609 platelets/μL (mean ± SD) was increased 151% ± 7% to 754 125 ± 3397 platelets/μL in the PRP preparation. Figure 1 represents a cranial CSD with each therapy. Histologic staining of cross-section slices of the cranium revealed that control defects healed with a combination of new bone growth and dense fibrous connective tissue (Figure 1). Magnified images of the control groups (Figure 2) demonstrated that new bone growth was mainly limited to the periphery of the defect. A collapse of the defect occurred between the PTFE membranes, creating a thin residual defect. The animals that received only PRP after CSD surgery appeared similar to control groups with bone growth mainly at the periphery and connective tissue throughout a collapsed defect (Figures 1 and 3).

Unlike the PRP group, CSDs containing only Bio-Oss particles demonstrated a greater amount of bone growth near the center of the defect (Figures 1 and 4). The groups grafted with Bio-Oss showed intimate contact between new bone and Bio-Oss particles, with new bone surrounding Bio-Oss particles and new bone internal to Bio-Oss particles. The borders of new bone areas showed osteoblastic rimming and evidence of osteoclastic activity. New bone growth was also found external to the membranes used to isolate the defects on both the side facing the dura (Figure 3) and the side facing the calvarial periosteum (Figure 4). Bio-Oss particles appeared largely intact, with little evidence of resorption, presenting space-maintaining characteristics.

The PRP plus Bio-Oss treatment did not appear to further enhance new bone formation as compared with Bio-Oss alone (Figures 1 and 5). The original space between the defect was maintained in both the Bio-Oss and PRP plus Bio-Oss groups. Also of note, there was no inflammatory cell response in any of the 4 groups that was indicative of graft rejection. However, it is important to note that the rats were all of the same genetic strain, which may account for the lack of an inflammatory response, even though the PRP preparations were pooled from multiple rats.

Radiographs of the calvarial defects yielded little information about regeneration of the defects via new bone growth because of the radiodense properties of the Bio-Oss particles and because the particles were not resorbed during the 8-week time of this study (Figure 6). Therefore, the groups grafted with Bio-Oss appeared densely filled, with Bio-Oss particles completely filling in the defect. The control group and PRP groups appeared radiolucent, with the defects still visible. However, some new bone regeneration was apparent in both groups at the periphery of the defect.

The purpose of this study was to assess the regenerative capacities of combination therapy with an anorganic bovine bone Bio-Oss and PRP, alone or in combination, nonspontaneously healing CSD to evaluate osseous wound healing. Qualitative comparisons were made among different groups via histologic and radiographic techniques. The CSDs from control and PRP groups healed in a very thin dimension, and the tissue consisted of new bone originating from the periphery of the defects. The centers of the defects were filled mainly with dense fibrous connective tissue. Space maintenance was absent in the control and PRP groups, indicating that the defect was not stable. In contrast to the control and PRP animals, treatment with Bio-Oss and PRP plus Bio-Oss after CSD resulted in greater stability within the membranes because of the space-maintaining characteristics of Bio-Oss. The pattern of new bone growth was also different in that Bio-Oss particles were well integrated into the new bone throughout the defect and in central locations; however, new bone formation was limited because of incomplete resorption of Bio-Oss in the central locations. Radiomorphometric findings confirmed histologic data that the control and PRP groups had a radiolucent appearance, with evidence of radiodensity representing new bone growth forming at the periphery of all defects. The Bio-Oss and the PRP plus Bio-Oss groups demonstrated uniformly radiodense granular appearances, owing to the presence of nonresorbed Bio-Oss particles. The characteristic radiodensity of Bio-Oss particles prevented a densitometric evaluation of these radiographs to assess new bone growth.

Placing PTFE membranes on either side of the calvarial defect is a technique that has been commonly used in this laboratory to isolate the defects.^13,14  Bio-Oss was used to provide a source of hydroxyapatite bone mineral, as well as a trabecular microstructure similar to human bone, creating an osteoconductive effect. Additionally, the incorporation of a solid particulate graft material ensures complete anatomic space maintenance. Linde et al¹⁵  refer to this technique as “osteopromotion,” referring to the use of physical means to seal off an anatomic site in order to prevent other tissues, notably connective tissue, from interfering with osteogenesis. In-growth of connective tissue and other rapidly regenerating tissues is prevented, allowing slower migrating cells with osteogenic potential to migrate into the area from the adjacent bone margins and to form new bone relatively undisturbed.

Resorption and replacement of the Bio-Oss particles may occur with time; however, the extent of resorption and length of time required is controversial.¹⁶  Still, these grafted sites have the potential to heal at a much greater thickness and strength than the control or PRP-only groups and have increased utility as bone graft materials in periodontics, where increased thickness and strength of new bone is necessary in sinus-lift procedures, alveolar ridge augmentation, or guided bone regeneration.

The PRP was tested as an autogenous source of growth factors recognized as promoting early bone regeneration (PDGF, IGF-I, and TGF-β), providing an osteoinductive effect. In the present study, CSDs from the control and PRP groups healed in a very thin dimension, and the tissue consisted of new bone originating from the periphery of the defects. Space maintenance was absent in the control and PRP groups, indicating that the defect was not stable. An important consideration for these findings is that even though animals were allowed to recover, the CSDs would most likely never heal completely. Previous studies have demonstrated that if allowed to mature for a longer period, these defects would not totally regenerate.¹² 

The 8-week course of the experiment was chosen to maximize assessment of the early effects of PRP addition, and this time frame may have greatly influenced the degree of maturity of the defects grafted with Bio-Oss. Several studies have shown excellent bone regeneration with Bio-Oss with more extended time periods for maturation of the grafted sites. Studies in dogs, primates, and humans have demonstrated remarkable bone regeneration at 6–7 months after implant surgery.^17–19  With an extended period of time to allow for graft maturation in this rat model (12 to 16 weeks), increased bone growth might occur in the defects grafted with Bio-Oss.

Artzi et al²⁰  evaluated Bio-Oss in its cancellous form when grafted in 15 fresh human extraction sockets in 15 patients. The study found the average clinical bone fill of the augmented sockets was 82.3%. New osseous tissue adhered to the Bio-Oss particles. Also, the resorbability of the porous bovine bone mineral could not be recognized in the 9-month period of the study. In a following study, histochemical analysis of the samples previously obtained from extraction sockets grafted with Bio-Oss demonstrated grafted particles that were not significantly resorbed at 9 months.²¹  The researchers concluded that cancellous anorganic bovine bone is a biocompatible filler agent in extraction socket sites and an acceptable graft for edentulous ridge preservation at sites prepared to receive endosseous implants. The Bio-Oss was determined to be osteoconductive based on the promotion of osseous ingrowth and close integration with newly generated bone. In other studies that examined the ability of Bio-Oss to induce new bone formation, investigators deproteinized cancellous bovine bone and applied chaotropic solvents used in the protocol for purification of bone morphogenic proteins.²²  The results showed that small amounts of protein were present in the Bio-Oss in close association with the mineral phase. Some of the extracted material possessed osteoinductive potential, and the presence of growth factors (TGF-β and bone morphogenic protein-2) were detected; which may explain the osteopromotive ability of Bio-Oss.

The current study was designed to evaluate potential additive or synergistic affects by combining Bio-Oss and PRP. The PRP may adsorb onto the surface of the Bio-Oss particles, rendering them osteoconductive. This concept is supported by Jiang et al,²³  in a study in which PDGF-BB and IGF-I were radiolabeled and incubated with anorganic bovine bone matrix. Results showed that PDGF-BB and IGF-I adsorbed to the bone mineral matrix in a concentration-dependent fashion. This suggests that these growth factors can be absorbed onto the anorganic bovine bone mineral matrix and may subsequently enhance the osteogenic properties of this bone graft material. In the present study, increased bone formation due to PRP therapy after CSD was not detected. Platelet counts in PRP resulted in approximately 754 000 platelets/μL, which was similar to the PRP platelet counts achieved by Marx et al.²  However, the mean percent increase in platelet concentration, 151%, was lower than that achieved in other reported studies. The coagulation of the PRP produced a gel that had excellent handling characteristics and could easily be placed within the calvarial defect. The fact that the PRP group did not regenerate bone in an amount greater than the control group contradicts earlier findings of greater bone density in bone grafts in which PRP was added.²  The control group may have performed similarly to PRP, as it contained a coagulum of whole blood, which formed a fibrin clot and would contain platelets that released growth factors (although at a lower concentration).

Kim et al²⁴  reported results similar to those in this study using demineralized bone powder plus PRP in a dog model. Histologic analysis showed that all of the bone defects surrounding implants and treated with demineralized bone powder, with or without PRP, were filled with new bone at 6 weeks, with enhanced bone maturity. Studies that evaluated the effect of PRP on bone healing of autogenous bone grafts in CSDs in rabbits did detect greater bone formation at earlier 4-week points but not at 12 weeks after surgery.^25,26  The researchers concluded that the beneficial effects of PRP were greatest at the initial healing period of 4 weeks, an observation supported by our pilot study at 6 weeks and our experiments at 8 weeks. In contrast, other studies observed a histologically significant enhancement of bone healing in PRP treated vs control rats, with an 8-mm CSD, at both 4 and 12 weeks,²⁷  while others reported that PRP soak loaded onto a absorbable collagen sponge did not enhance bone regeneration in rats at 4 or 8 weeks vs sponge or controls in a 6-mm CSD model.²⁸  Subsequent radiographic examination in the latter study suggested that the PRP preparation did not have a significant effect on osteogenesis.²⁹ 

Mariano et al³⁰  evaluated the ability of PRP to heal a CSD in diabetic rats at 30 days. Control rats exhibited newly formed bone close to the borders of the surgical defect, while the PRP-treated rats exhibited a greater amount of newly formed bone close to the borders of the surgical defect, with regenerating bone often bridging the defect. These conflicting observations, including our own, may be at least partially explained by the findings of Nagata et al,³¹  who observed that bone healing in rat CSDs is influenced by the proportion of graft material to PRP. The variability of results seen in clinical and animal studies may have several explanations. A standardized PRP preparation and the addition of a titanium mesh for space maintenance may help resolve these discrepancies and guide clinicians in the use of PRP in clinical settings.

It can be concluded from this study that all groups (control [hemotologic coagulum], PRP, Bio-Oss, and Bio-Oss plus PRP), produced osseous regeneration when grafted into a CSD within the rat calvarium and allowed to heal for 8 weeks. Areas grafted with Bio-Oss had a greater space-maintaining capacity and the potential to heal in a greater dimension. It is also apparent from these studies that any positive effects of PRP on the healing process could not be determined at 8 weeks. The possibility that early positive effects of PRP-derived growth factors occurred before being naturally degraded suggests that their effects on osseous regeneration were short lived or not significant. However, PRP has handling characteristics, that are advantageous for use with grafting materials as it is gelatinous and comes from an autologous source. This suggests that PRP can serve as an excellent vehicle for placing and handling particulate bone graft materials, which may be of significant benefit.

Abbreviations

CSD

critical-size defect

IGF-I, IGF-II

insulin-like growth factors

PDGF

platelet-derived growth factor

PRP

platelet-rich plasma

PTFE

polytetrafluoroethylene

TGF-β

transforming growth factor beta

The opinions expressed in this article are exclusively those of the authors and not necessarily those of the US Army Dental Corps or the Department of the Army. The use of commercial products in this project does not imply endorsement by the US government.