Effects of pamidronate on human alveolar osteoblasts in vitro.

PURPOSE: Administration of bisphosphonates has recently been associated with the development of osteonecrotic lesions of the jaw (ONJ). To elucidate the potential contributions of osteogenic cells to the development and regeneration of ONJ, we have isolated primary cells from human alveolar and long/iliac bones, and examined the effects of pamidronate on cell viability, proliferation, osteogenesis, and wound healing. MATERIALS AND METHODS: Primary human osteoblasts and bone marrow stromal cells were isolated from alveolar and iliac/long bone and marrow tissue. Cellular proliferation, alkaline phosphatase activity, apoptosis (terminal deoxynucleotidyl transferase dUTP nick end labeling, caspase-3, and 4,6-diamidino-2-phenylindole dihydrochloride assays) and wound healing in an in vitro scratch assay were assessed after exposure to pamidronate at a range of clinically relevant doses. RESULTS: Primary alveolar osteoblasts proliferated at significantly higher rates than long/iliac bone osteoblasts in vitro. Upon exposure of alveolar osteoblasts and long/iliac bone marrow stromal cells to pamidronate for more than 72 hours, we have observed significantly decreased cell viability, proliferation, osteogenesis, and in vitro wound healing at ≥6 × 10(-5) mol/L pamidronate, with the induction of apoptosis in approximately 20% of cell population. CONCLUSIONS: The remodeling activity of alveolar bone, indicated by higher proliferation of alveolar osteoblasts, could be negatively affected by exposure to high concentrations of pamidronate over extended periods. The absence of anabolic effects of pamidronate on alveolar osteoblasts and the induction of apoptosis in osteogenic cells could negatively affect bone balance at this site and contribute to osteonecrosis of the jaw.

Novel therapy to reverse the cellular effects of bisphosphonates on primary human oral fibroblasts.

PURPOSE: Osteonecrosis of the jaws (ONJ) is a clinical condition that is characterized by a nonhealing breach in the oral mucosa resulting in exposure of bone and has been increasingly reported in patients receiving bisphosphonate (BP) therapy. Although the pathogenesis and natural history of ONJ remain ill-defined, it appears that the oral soft tissues play a critical role in the development of this condition. We examined the effects of the nitrogen-containing BPs pamidronate and zoledronate on primary human gingival fibroblasts. MATERIALS AND METHODS: Primary gingival fibroblasts were exposed to clinically relevant doses of pamidronate and zoledronate. Cellular proliferation was measured with an MTS/PMS reagent-based kit (Promega, Madison, WI), scratch wound assays were performed to measure cellular migration, and apoptosis was measured by use of terminal deoxynucleotidyl transferase-mediated dUTP-FITC end labeling and caspase assays. The BP-exposed cells were treated with 10-ng/mL recombinant human platelet-derived growth factor BB (rhPDGF-BB) and 50-µmol/L geranylgeraniol (GGOH). RESULTS: Gingival fibroblasts are significantly more sensitive to inhibition of proliferation by zoledronate compared with pamidronate. Exposure of these cells to pamidronate but not zoledronate resulted in an increase in cellular apoptosis. Furthermore, exposure of gingival fibroblasts to pamidronate or zoledronate resulted in a decrease in cellular migration. We show that these defects are due to a loss of cell-substratum adhesion and a reduction of F-actin bundles. Finally, we show that the addition of rhPDGF-BB and GGOH in vitro is able to partially rescue the cell proliferation, migration, and adhesion defects. CONCLUSION: The cytotoxic effects of BPs on oral fibroblasts and their significant reversal by the addition of GGOH and rhPDGF-BB provide both the potential mechanism and treatment options for ONJ.

Alternative indications for bisphosphonate therapy.

Bisphosphonates are currently used in the treatment of osteoporosis (postmenopausal and steroid-induced), hypercalcemia of malignancy, Paget's disease of bone, multiple myeloma, and skeletally related events associated with metastatic bone disease in breast, prostate, lung, and other cancers. There are, however, numerous other conditions where a decrease in bone remodeling by bisphosphonates might aid in disease management. The focus of this review will be to discuss a select group of conditions for which bisphosphonate therapy may be efficacious. In this review we present several cases where bisphosphonates have been used as a primary or adjunctive treatment for giant cell lesions of the jaws. Use of bisphosphonate therapy for giant cell tumors of the appendicular skeleton, pediatric osteogenesis imperfecta, fibrous dysplasia, Gaucher's disease, and osteomyelitis will be discussed. Finally, we will review previous in vivo studies on the use of bisphosphonates to augment integration and to treat osteolysis surrounding failing orthopedic implants.

Osteonecrosis of the jaw and bisphosphonate treatment for osteoporosis.

A potential side effect associated with bisphosphonates, a class of drugs used in the treatment of osteoporosis, Paget's disease and metastatic bone disease, is osteonecrosis of the jaw (ONJ). The incidence of ONJ in the general population is unknown; this rare condition also may occur in patients not receiving bisphosphonates. Case reports have discussed ONJ development in patients with multiple myeloma or metastatic breast cancer receiving bisphosphonates as palliation for bone metastases. These patients are also receiving chemotherapeutic agents that might impair the immune system and affect angiogenesis. The incidence or prevalence of ONJ in patients taking bisphosphonates for osteoporosis seems to be very rare. No causative relationship has been unequivocally demonstrated between ONJ and bisphosphonate therapy. A majority of ONJ occurs after tooth extraction. Furthermore, the underlying risk of developing ONJ may be increased in osteoporotic patients by comorbid diseases. Treatment for ONJ is generally conservative.

Inhibition of oral mucosal cell wound healing by bisphosphonates.

PURPOSE: Bisphosphonates (BPs) are a widely used class of drugs that are effective in the treatment and prevention of osteoporosis, hypercalcemia of malignancy, and bone metastases associated with multiple myeloma, breast cancer, and other solid tumors. In the past several years there have been numerous reports describing the occurrence of osteonecrosis of the jaws (ONJ) associated with these drugs. Whether the ONJ lesion initiates in the oral mucosa or derives from the underlying bone is not well understood. In this report we describe the effect of pamidronate, a second-generation BP, on oral mucosal cells. MATERIALS AND METHODS: Murine oral keratinocytes were isolated and exposed to pamidronate at a range of clinically relevant doses. Cellular proliferation was measured using a MTS/PMS reagent-based kit and wound healing was examined with a scratch assay. To determine whether oral keratinocytes undergo apoptosis following exposure to pamidronate, TUNEL, caspase-3, and DAPI apoptosis assays were performed. RESULTS: We show that BP pretreatment of oral mucosal cells inhibits proliferation and wound healing at clinically relevant doses, and that this inhibition is not due to cellular apoptosis. CONCLUSIONS: To our knowledge this is the first report investigating the effect of nitrogen-containing BPs on oral mucosal cells. This study suggests that BPs inhibit oral keratinocyte wound healing which may play a significant role in the initiation of ONJ.

Incidence of osteonecrosis of the jaw in women with postmenopausal osteoporosis in the health outcomes and reduced incidence with zoledronic acid once yearly pivotal fracture trial.

BACKGROUND: The authors determined incidence of osteonecrosis of the jaw (ONJ) in a large, prospective three-year clinical trial of zoledronic acid in women with postmenopausal osteoporosis (PMO). METHODS: A total of 7,714 women with PMO received intravenous zoledronic acid 5 mg or a placebo. No spontaneous reports of ONJ were received. An independent, blinded adjudication committee searched the trial's adverse event database by using 60 terms. On an ongoing basis, the committee reviewed the identified events, and it defined ONJ as exposed bone in the maxillofacial area with delayed healing for more than six weeks despite appropriate care. RESULTS: One participant who received a placebo and one participant who received zoledronic acid experienced delayed healing associated with infection. Both conditions resolved after antibiotic therapy, débridement or both. CONCLUSION: The occurrence of ONJ is rare in a PMO population, and delayed healing of lesions can occur with and without bisphosphonate use over three years. CLINICAL IMPLICATIONS: The low incidence of ONJ must be assessed in the context of the clinical benefit of zoledronic acid therapy in reducing hip, vertebral and nonvertebral fractures in this at-risk population. There is no evidence to suggest that healthy patients with osteoporosis who are receiving bisphosphonates require any special treatment beyond routine dental care or to support altering standard treatment practices.

Bisphosphonate-associated osteonecrosis of the jaw: conclusions based on an analysis of case series.

ONJ appears to be associated with BPs; however, the pathophysiology, incidence, and co-morbidities require further investigation. The major risk factors identified to date appear to be cancer (or chemotherapy for cancer) and dental procedures or oral trauma. A clear definition of ONJ is critical to understanding this disease entity. Although recommendations regarding the prevention and management of ONJ exist, clinical studies are needed to establish more definitive guidelines for the management of ONJ. The use of intensive hyperbaric oxygen therapy may be beneficial to patients with ONJ.

Blood Products: What Oral and Maxillofacial Surgeons Need to Know.

Blood products are routinely used to manage various coagulation and hematological disorders. However, there is a debate in the medical literature concerning the appropriate use of blood and blood products. Oral and maxillofacial surgeons must have a basic knowledge and understanding of the various available products. A consultation with each patient's hematologist is always advised in order to decrease the risk of adverse events and improve the patient's safety.

Potential pathophysiological mechanisms in osteonecrosis of the jaw.

Bisphosphonates are used in the treatment of hypercalcemia of malignancy, skeletal complications associated with metastastic bone disease, Paget's disease, and osteoporosis. Osteonecrosis of the jaw (ONJ) is a recently described clinical condition that has been associated with the use of nitrogen-containing bisphosphonates. Reports describing this entity first appeared in the literature in 2003. While there have been significant numbers of case reports and a limited number of retrospective and prospective studies examining risk factors associated with ONJ, the pathophysiology of this condition remains elusive. In this review, we explore proposed mechanisms underlying ONJ development and identify potential areas for future investigation.

Osteonecrosis of the jaw and the role of bisphosphonates: a critical review.

Osteonecrosis of the jaw (ONJ), a condition characterized by necrotic exposed bone in the maxillofacial region, has been reported in patients with cancer receiving bisphosphonate therapy, and rarely in patients with postmenopausal osteoporosis or Paget disease of bone receiving such therapy. In the absence of a uniform definition, the American Academy of Oral and Maxillofacial Surgeons (AAOMS), the American Society for Bone and Mineral Research (ASBMR), and other groups have established similar diagnostic criteria for bisphosphonate-related ONJ, which is more commonly reported in patients with advanced malignancies with skeletal metastases who receive higher doses, and is more rarely reported in patients with osteoporosis and Paget disease who receive lower doses. However, a critical review of the literature reveals that the etiology of ONJ remains unknown, and to date no direct causal link to bisphosphonates has been established. Despite an increased awareness of ONJ and recent improvements in preventive strategies, patients and physicians alike continue to express concern about the potential risks of bisphosphonate treatment in both oncologic and nononcologic settings. Although much remains to be learned about this condition, including its true incidence in various patient populations, its pathophysiology, and optimal clinical management, evidence to date suggests that the positive benefits of bisphosphonates in patients with malignant bone disease, osteoporosis, or Paget disease outweigh the relatively small risk of ONJ.

Phosphaturic mesenchymal tumor, mixed connective tissue variant, of the mandible: report of a case and review of the literature.

Tumor-induced osteomalacia (TIO) is a rare paraneoplastic syndrome that results in renal phosphate wasting with hypophosphatemia. In most cases, the underlying cause of TIO is a small mesenchymal neoplasm that is often difficult to detect, resulting in delayed diagnosis. One such neoplasm is the phosphaturic mesenchymal tumor, mixed connective tissue variant (PMTMCT), an unusual entity with unique morphologic and biochemical features. Most of these tumors are found at appendicular sites with only rare cases reported in the jaws. We describe a PMTMCT involving the mandible in a patient with a protracted history of osteomalacia. A review of the current literature is provided with emphasis on the clinical and histologic features, etiopathogenesis, and management of PMTMCT in the setting of TIO.

The expression of the receptor for glycation endproducts (RAGE) in oral squamous cell carcinomas.

Advanced glycation endproducts (AGEs) and their receptors, receptor for advanced glycation endproducts (RAGE), are novel groups of molecules with roles in inflammation, cytokine activation, and promotion of cell growth. Recently, RAGE has been implicated in the progression and metastasis of several epithelial tumors. The expression of RAGE was examined in 38 oral squamous cell carcinoma (OSCC) cases by immunohistochemistry. In the OSCCs, RAGE positivity, interpreted as more than 25% positive cells, was detected in 10 of 10 well-differentiated, 3 of 4 well-to-moderately differentiated, 3 of 9 moderately differentiated, 1 of 7 moderate-to-poorly differentiated, and 0 of 8 poorly differentiated tumors. The staining percentage was significantly higher in well-differentiated tumors compared to moderately (P < .05) and poorly differentiated (P < .05) tumors. All normal mucosa samples were RAGE-positive. Western blot analysis for RAGE was performed on 2 OSCCs and 2 normal oral mucosa samples. Higher expression was observed in the normal tissues compared to the OSCCs. Our results show that RAGE immunoreactivity correlates with histologic differentiation in OSCC.

Controlled delivery of platelet-rich plasma-derived growth factors for bone formation.

Platelet-rich plasma (PRP) represents an autologous source of growth factors essential for bone regeneration. The clinical efficacy of PRP is, however, unpredictable, and this is likely due to the inefficient and inconsistent delivery of PRP-derived growth factors. Previous investigations have shown that current methods of PRP preparation result in a premature release of the relevant bone stimulatory factors. As successful bone regeneration requires multiple factors presented in a physiologic temporal and spatial cascade, the objective of this study is to control the bioavailability of PRP-derived growth factors using a hydrogel carrier system. Specifically, the release of platelet-derived growth factor, transforming growth factor beta-1, and insulin-like growth factor from two types of alginate carriers was compared over time. The effects of the released factors on the growth and alkaline phosphatase (ALP) activity of human osteoblast-like cells were also evaluated. It was found that factor release profiles varied as function of carrier type, and binding of growth factors to the alginate matrix also modulated their release. The bioactivity of released factors was maintained in vitro and they promoted cell proliferation and ALP activity. These results demonstrate the potential of this autologous multifactor delivery system for controlling the bioavailability of PRP-derived factors. Future studies will focus on optimizing this system to increase the clinical efficacy of PRP by matching the distribution and temporal sequencing of PRP-derived factors to the bone healing cascade.

Controlled release of PRP-derived growth factors promotes osteogenic differentiation of human mesenchymal stem cells.

Platelet-rich plasma (PRP) has been gaining increasing popularity in orthopedics and oral and maxillofacial surgery because of its potential efficacy in enhancing bone regeneration. To maximally augment bone healing using PRP and to control the bioavailability of the relevant growth factors, we have designed an alginate hydrogel-based PRP-delivery system. The bioactivity of the growth factors released from PRP carriers was evaluated by determining the ability of these factors to induce osteogenic differentiation of human mesenchymal stem cells (hMSCs). Specifically, monolayers of hMSCs were incubated with the PRP-containing hydrogel carriers over a two-week culture period. Osteoblast-like cells treated with the hydrogel carriers served as controls. The growth and osteogenic differentiation (alkaline phosphatase activity and mineralization) of hMSCs was determined. The results showed that PRP-derived growth factors released from hydrogel carriers stimulated the osteogenic differentiation of hMSCs and most significantly, the cellular response was carrier type-dependent. Future studies will focus on in vitro and in vivo testing of the efficacy of hydrogel-based PRP release systems.

Differential growth factor retention by platelet-rich plasma composites.

PURPOSE: This study evaluates the temporal sequence and growth factor release from platelet-rich plasma (PRP) combined with different bone substitutes (BS), to identify an optimal substrate for extended growth factor retention. MATERIALS AND METHODS: PRP was clotted with bovine thrombin or thrombin receptor activator peptide-6 (TRAP). In addition, PRP was clotted using Allogro (Ceramed, Lakewood, CO), BioGlass (Mo-Sci, Rolla, MN), or BioOss (Osteohealth, Shirley, NY). The effects of media exchange and BS on platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF beta) release were quantified via enzyme-linked immunosorbent assay. RESULTS: At day 1, the thrombin group released 36% more PDGF than the TRAP group and 80% more than the BS groups. At 7 days, PDGF release was the greatest for the TRAP group. PDGF release was minimal for all groups at day 14, with BS groups retaining 60% more PDGF than thrombin clots. Similarly, the thrombin group released the greatest amount of TGF beta (81.4% of the total), whereas TRAP and BS groups released significantly less TGF beta at day 1. Compared with thrombin, TRAP retained 39.2% more TGF beta, whereas BS groups retained even greater levels (Allogro, 54.3%; BioOss, 45.8%; BioGlass, 67.0%). No significant difference in TGF beta release was observed among the substitutes after day 1. The BS groups continued to retain TGF beta after 14 days, whereas all TGF beta in the thrombin clots was depleted. CONCLUSIONS: PRP preparation with thrombin results in a large, immediate release of growth factors that could be lost into the interstitium in vivo. TRAP-BS may prove more efficacious than thrombin in sustaining growth factor levels critical for the cascade of events leading to bone formation.

Activation of platelet-rich plasma using thrombin receptor agonist peptide.

PURPOSE: This study proposes an alternative preparation method of platelet-rich plasma (PRP). Specifically, we compare the use of thrombin receptor agonist peptide-6 (TRAP) and bovine thrombin as a clotting agent in the preparation of PRP. MATERIALS AND METHODS: PRP was prepared by centrifugation and clotted with thrombin or TRAP. In vitro clotting times were monitored as a function of TRAP concentration, and clot retraction was determined by measuring clot diameter over time. Following the optimization of TRAP concentration, experiments were repeated with the addition of several commercially available bone substitutes. The release of PRP-relevant growth factors as a function of PRP preparation was also determined. RESULTS: The most rapid polymerization of PRP takes place with the addition of thrombin, followed by TRAP/Allogro (Ceramed, Lakewood, CO), TRAP/BioGlass (Mo-Sci, Rolla, MN), TRAP/BioOss (Osteohealth, Shirley, NY), and TRAP alone. Thrombin caused considerable clot retraction (43%), whereas TRAP alone resulted in only 15% retraction. TRAP/Allogro, TRAP/BioOss, and TRAP/BioGlass all exhibited minimal retraction (8%). CONCLUSIONS: The use of TRAP to activate clot formation in the preparation of PRP may be a safe alternative to bovine thrombin. It results in an excellent working time and significantly less clot retraction than the currently available methods of PRP production.