Osteoinduction and osteoimmunology: Emerging concepts.

The recognition and importance of immune cells during bone regeneration, including around bone biomaterials, has led to the development of an entire field termed "osteoimmunology," which focuses on the connection and interplay between the skeletal system and immune cells. Most studies have focused on the "osteogenic" capacity of various types of bone biomaterials, and much less focus has been placed on immune cells despite being the first cell type in contact with implantable devices. Thus, the amount of literature generated to date on this topic makes it challenging to extract needed information. This review article serves as a guide highlighting advancements made in the field of osteoimmunology emphasizing the role of the osteoimmunomodulatory properties of biomaterials and their impact on osteoinduction. First, the various immune cell types involved in bone biomaterial integration are discussed, including the prominent role of osteal macrophages (OsteoMacs) during bone regeneration. Thereafter, key biomaterial properties, including topography, wettability, surface charge, and adsorption of cytokines, growth factors, ions, and other bioactive molecules, are discussed in terms of their impact on immune responses. These findings highlight and recognize the importance of the immune system and osteoimmunology, leading to a shift in the traditional models used to understand and evaluate biomaterials for bone regeneration.

Histological evaluation following treatment of recession-type defects with coronally advanced flap and a novel human recombinant amelogenin.

OBJECTIVES: To histologically evaluate the effects of a novel human recombinant amelogenin (rAmelX) on periodontal wound healing / regeneration in recession-type defects. MATERIALS AND METHODS: A total of 17 gingival recession-type defects were surgically created in the maxilla of three minipigs. The defects were randomly treated with a coronally advanced flap (CAF) and either rAmelX (test), or a CAF and placebo (control). At three months following reconstructive surgery, the animals were euthanized, and the healing outcomes histologically evaluated. RESULTS: The test group yielded statistically significantly (p = 0.047) greater formation of cementum with inserting collagen fibers compared with the control group (i.e., 4.38 mm ± 0.36 mm vs. 3.48 mm ± 1.13 mm). Bone formation measured 2.15 mm ± 0.8 mm in the test group and 2.24 mm ± 1.23 mm in the control group, respectively, without a statistically significant difference (p = 0.94). CONCLUSIONS: The present data have provided for the first-time evidence for the potential of rAmelX to promote regeneration of periodontal ligament and root cementum in recession-type defects, thus warranting further preclinical and clinical testing. CLINICAL RELEVANCE: The present results set the basis for the potential clinical application of rAmelX in reconstructive periodontal surgery.

Evidence of re-osseointegration after electrolytic cleaning and regenerative therapy of peri-implantitis in humans: a case report with four implants.

OBJECTIVE: To evaluate re-osseointegration after electrolytic cleaning and regenerative therapy of dental implants with peri-implantitis in humans. MATERIAL AND METHODS: Four dental implants that developed peri-implantitis underwent electrolytic cleaning followed by regenerative therapy with guided bone regeneration. All four implants developed recurrent peri-implantitis and were therefore explanted 6 to 13 months later. Radiographic bone level, probing depth, and bleeding on probing were determined at the time of surgery, 6 months later, and before implant retrieval. The peri-implant tissues were histologically and histomorphometrically analyzed. RESULTS: All four implants demonstrated radiographic and histological bone gain, reduced probing depth, and bleeding on probing. Radiographic bone gain was 5.8 mm mesially and 4.8 mm distally for implant #1, 3.3 mm and 2.3 mm for implant #2, 3.1 mm and 0.5 mm for implant #3, and 3.5 mm and 2.8 mm for implant #4. The histometric mean and maximum vertical bone gain for implant #1 to #4 was 1.65 mm and 2.54 mm, 3.04 mm and 3.47 mm, 0.43 mm and 1.27 mm, and 4.16 mm and 5.22 mm, respectively. The percentage of re-osseointegration for implant #1 to #4 was 21.0%, 36.9%, 5.7%, and 39.0%, respectively. In one implant, the newly formed bone was deposited directly onto calculus on the implant surface. CONCLUSIONS: We found that (1) re-osseointegration is possible on a formerly contaminated implant surface and (2) the electrolytic cleaning process seems to be effective enough at sites with calculus residues. CLINICAL RELEVANCE: Since re-osseointegration can be achieved by electrolytic cleaning, this decontamination technique may be considered as a future treatment concept.

Cross-linked hyaluronic acid slows down collagen membrane resorption in diabetic rats through reducing the number of macrophages.

OBJECTIVES: We previously showed that accelerated degradation of collagen membranes (CMs) in diabetic rats is associated with increased infiltration of macrophages and blood vessels. Since pre-implantation immersion of CMs in cross-linked high molecular weight hyaluronic acid (CLHA) delays membrane degradation, we evaluated here its effect on the number of macrophages and endothelial cells (ECs) within the CM as a possible mechanism for inhibition of CM resorption. MATERIALS AND METHODS: Diabetes was induced with streptozotocin in 16 rats, while 16 healthy rats served as control. CM discs were labeled with biotin, soaked in CLHA or PBS, and implanted under the scalp. Fourteen days later, CMs were embedded in paraffin and the number of macrophages and ECs within the CMs was determined using antibodies against CD68 and transglutaminase II, respectively. RESULTS: Diabetes increased the number of macrophages and ECs within the CMs (∼2.5-fold and fourfold, respectively). Immersion of CMs in CLHA statistically significantly reduced the number of macrophages (p < 0.0001) in diabetic rats, but not that of ECs. In the healthy group, CLHA had no significant effect on the number of either cells. Higher residual collagen area and membrane thickness in CLHA-treated CMs in diabetic animals were significantly correlated with reduced number of macrophages but not ECs. CONCLUSIONS: Immersion of CM in CLHA inhibits macrophage infiltration and reduces CM degradation in diabetic animals. CLINICAL RELEVANCE: The combination of CLHA and CM may represent a valuable approach when guided tissue regeneration or guided bone regeneration procedures are performed in diabetic patients.

Conventional finite element models estimate the strength of metastatic human vertebrae despite alterations of the bone's tissue and structure.

INTRODUCTION: Pathologic vertebral fractures are a major clinical concern in the management of cancer patients with metastatic spine disease. These fractures are a direct consequence of the effect of bone metastases on the anatomy and structure of the vertebral bone. The goals of this study were twofold. First, we evaluated the effect of lytic, blastic and mixed (both lytic and blastic) metastases on the bone structure, on its material properties, and on the overall vertebral strength. Second, we tested the ability of bone mineral content (BMC) measurements and standard FE methodologies to predict the strength of real metastatic vertebral bodies. METHODS: Fifty-seven vertebral bodies from eleven cadaver spines containing lytic, blastic, and mixed metastatic lesions from donors with breast, esophageal, kidney, lung, or prostate cancer were scanned using micro-computed tomography (µCT). Based on radiographic review, twelve vertebrae were selected for nanoindentation testing, while the remaining forty-five vertebrae were used for assessing their compressive strength. The µCT reconstruction was exploited to measure the vertebral BMC and to establish two finite element models. 1) a micro finite element (µFE) model derived at an image resolution of 24.5 µm and 2) homogenized FE (hFE) model derived at a resolution of 0.98 mm. Statistical analyses were conducted to measure the effect of the bone metastases on BV/TV, indentation modulus (Eit), ratio of plastic/total work (WPl/Wtot), and in vitro vertebral strength (Fexp). The predictive value of BMC, µFE stiffness, and hFE strength were evaluated against the in vitro measurements. RESULTS: Blastic vertebral bodies exhibit significantly higher BV/TV compared to the mixed (p = 0.0205) and lytic (p = 0.0216) vertebral bodies. No significant differences were found between lytic and mixed vertebrae (p = 0.7584). Blastic bone tissue exhibited a 5.8% lower median Eit (p< 0.001) and a 3.3% lower median Wpl/Wtot (p<0.001) compared to non-involved bone tissue. No significant differences were measured between lytic and non-involved bone tissues. Fexp ranged from 1.9 to 13.8 kN, was strongly associated with hFE strength (R2=0.78, p< 0.001) and moderately associated with BMC (R2=0.66, p< 0.001) and µFE stiffness (R2=0.66, p< 0.001), independently of the lesion type. DISCUSSION: Our findings show that tumour-induced osteoblastic metastases lead to slightly, but significantly lower bone tissue properties compared to controls, while osteolytic lesions appear to have a negligible impact. These effects may be attributed to the lower mineralization and woven nature of bone forming in blastic lesions whilst the material properties of bone in osteolytic vertebrae appeared little changed. The moderate association between BMC- and FE-based predictions to fracture strength suggest that vertebral strength is affected by the changes of bone mass induced by the metastatic lesions, rather than altered tissue properties. In a broader context, standard hFE approaches generated from CTs at clinical resolution are robust to the lesion type when predicting vertebral strength. These findings open the door for the development of FE-based prediction tools that overcomes the limitations of BMC in accounting for shape and size of the metastatic lesions. Such tools may help clinicians to decide whether a patient needs the prophylactic fixation of an impending fracture.

Hyaluronic acid slows down collagen membrane degradation in uncontrolled diabetic rats.

AIM: To examine the in vitro biokinetics of hyaluronic acid (HA) from a collagen membrane (CM) and to evaluate the in vivo effect of immersion of the CM in HA solution on its degradation in streptozotocin (STZ)-induced diabetes conditions in a rat calvaria subcutaneous model. BACKGROUND: CM degradation is accelerated in uncontrolled diabetic rats. Immersion of CM in HA has been suggested to decrease their resorption rate without interfering with their tissue integration and structural degradation. However, it is unknown to what extent CM degradation may be influenced by its immersion in HA solution under a condition mimicking a medically compromised situation with an increased inflammatory level such as diabetes. MATERIALS AND METHODS: CMs were soaked in cross-linked HA. Protein adsorption and the HA release were quantified by ELISA. Diabetes was induced in sixteen rats, while 16 healthy rats served as control. CM was prepared and labeled prior to implantation with Biotin. Seventeen CM were immersed in HA and 17 CM in PBS. In each animal, one test or one control disk was implanted. In order to compare the collagen content, two similar non-implanted CM were used as baseline. Fourteen days after surgery, thirty-two animals were sacrificed. The entire calvaria including the skin above, was chemically fixed, decalcified, and embedded in paraffin. Five-µm-thick sections were analyzed histologically and histomorphometrically using H&E and avidin-peroxidase staining. RESULTS: The in vitro results demonstrated that the CM adsorbed roughly 80% of the total HA content. After 10 days, 36.3% of the initial HA remained on the CM. The in vivo results demonstrated that diabetes significantly reduced the thickness of the CM, while HA had a significant effect on keeping the membrane thickness. HA increased the residual collagen content in the diabetic group (P < 0.0001) but no such effect was observed in the healthy group. CONCLUSION: Immersion of CM in HA prior to the implantation delays membrane degradation in uncontrolled diabetic compared with normoglycemic rats.

Self-regenerative capacity of intra-oral bone defects.

OBJECTIVE: To provide an overview on the self-regenerative capacity of various types of intra-oral bone defects. MATERIALS AND METHODS: This paper has narratively reviewed the most important aspects of bone biology and the healing outcomes related to the self-regenerative capacity (i.e. without the placement of any biomaterial) of bone defects that occur following tooth extraction, autogenous graft harvesting, periapical lesions, cystic lesions of the jaws, third molar extraction and experimentally created ridge defects. RESULTS: In animals (i.e. dogs and monkeys), the greatest changes in horizontal and vertical dimension occur during the first 6 months following tooth extraction. In humans, bone remodelling may take from several months to years and exhibits marked inter-individual variability. Following tooth extraction at compromised sites (e.g. presence of severe bone loss at the time-point of extraction), the healing may occur slower and a substantial volume reduction can be expected than following tooth extraction at non-compromised sites. In the mandibular symphysis and ramus, the bone defects resulting following bone block harvesting are gradually healing to a large extent, but complete healing appears not to occur due to poorer space provision and wound stability capacities. Defects after peri-apical surgery display a substantial self-regenerative capacity and heal at a great extent without the use of any adjunct measures. The vast majority of jawbone defects after cystectomy heal at a great extent and without apparent influence in the shape of the jaw, without the need of adjunct measures. After surgical removal of mandibular third molars, bone fill can be observed over a period of at least 12 months, with the most substantial change (e.g. the greatest bone fill) occurring during the first 3 months after surgery. However, complete fill of these residual bone defects does not always occur. CONCLUSIONS: Intra-oral bone defects possess a high self-regenerative capacity. Factors such as extent of bone loss, presence of bony walls, closed healing environment, space provision and mechanical wound stability substantially influence healing/regeneration.

Macrophage behavior and interplay with gingival fibroblasts cultured on six commercially available titanium, zirconium, and titanium-zirconium dental implants.

OBJECTIVES: The host-material interface has been a crucial relationship dictating the successful integration of biomaterials, including dental implants. The aim of the present study was to first investigate how macrophages behaved on various dental implant surfaces and thereafter to investigate their effect on soft tissue cells. MATERIALS AND METHODS: Macrophage adhesion, proliferation, and polarization towards either an M1 or M2 phenotype were investigated on six implant surfaces fabricated from pure titanium (Ti), pure zirconium (ZLA), and a titanium-zirconium (Ti-Zi) alloy of various surface topographies/chemistries. Thereafter, conditioned media (CM) collected from macrophages seeded on these various implant surfaces was cultured with murine gingival fibroblasts and investigated for their ability to promote collagen synthesis. RESULTS: Macrophages attached and proliferated in similar levels on all implant surfaces; however, the modSLA hydrophilic surfaces tended to decrease the pro-inflammatory response by lowering the gene expression of TNF-alpha, IL-1, and IL-6 and promoting tissue resolution through the expression of an M2-macrophage cytokine IL-10. Thereafter, CM from macrophages were seeded with gingival fibroblasts on each implant surface. In general, CM from macrophages significantly promoted gingival fibroblast cell attachment on all implant surfaces at either 4 or 8 h and, most notably, significantly promoted fibronectin and TGF-beta gene expression on both Ti and Ti-Zi hydrophilic surfaces. CONCLUSIONS AND CLINICAL RELEVANCE: The present study found that implant surface topography and chemistry substantially impacted macrophage behavior. Most notably, modifications via hydrophilicity to both the pure Ti and Ti-Zi were shown to favor the secretion of macrophage pro-resolution markers and favored subsequent gingival fibroblast cell behavior when cultured with CM, whereas surface composition (Ti vs ZLA vs Ti-Zi) had little effect on macrophage polarization or gingival fibroblast behavior. This finding suggests that surface hydrophilicity would improve the soft tissue integration of dental implants, irrespective of material composition.

20-Year Follow-up in Maxillary Sinus Floor Elevation Using Bovine-Derived Bone Mineral: A Case Report with Histologic and Histomorphometric Evaluation.

Bovine-derived bone mineral demonstrated good osteoconductive properties as grating material for maxillary sinus floor elevation, but the long-term behavior of this material has not been reported. The purpose of this report was to analyze and compare histomorphometric measurements of new bone, bone graft, and medullar spaces 6 months, 12 months, and 20 years after grafting. In the grafted area, the amount of mineralized bone was 16.96% at 6 months, 22.53% at 12 months, and 22.05% at 20 years, respectively. The amount of bovine-derived bone mineral ranged from 35.87% to 4.85% in the same period. The volume of the newly formed mineralized bone does not increase over time, conversely to nonmineralized bone.

The periodontal pocket: pathogenesis, histopathology and consequences.

The conversion of junctional epithelium to pocket epithelium is regarded as a hallmark in the development of periodontitis. Knowledge of factors contributing to the initiation and progression of pocket formation is important and may result in the development of better preventive measures and improve healing outcomes after therapeutic interventions. The periodontal pocket is a pathologically deepened gingival sulcus. In healthy periodontal conditions, the defense mechanisms are generally sufficient to control the constant microbiological challenge through a normally functioning junctional epithelium and the concentrated powerful mass of inflammatory and immune cells and macromolecules transmigrating through this epithelium. In contrast, destruction of the structural integrity of the junctional epithelium, which includes disruption of cell-to-cell contacts and detachment from the tooth surface, consequently leading to pocket formation, disequilibrates this delicate defense system. Deepening of the pocket apically, and also horizontal expansion of the biofilm on the tooth root, puts this system to a grueling test. There is no more this powerful concentration of defense cells and macromolecules that are discharged at the sulcus bottom and that face a relatively small biofilm surface in the gingival sulcus. In a pocket situation, the defense cells and the macromolecules are directly discharged into the periodontal pocket and the majority of epithelial cells directly face the biofilm. The thinning of the epithelium and its ulceration increase the chance for invasion of microorganisms and their products into the soft connective tissue and this aggravates the situation. Depending on the severity and duration of disease, a vicious circle may develop in the pocket environment, which is difficult or impossible to break without therapeutic intervention.

The role of macrophage polarization on fibroblast behavior-an in vitro investigation on titanium surfaces.

OBJECTIVES: This study investigated the effect of smooth and rough titanium surface topographies on macrophage polarization and their influence on gingival fibroblast behavior cultured on titanium surfaces. MATERIALS AND METHODS: RAW 264.7 macrophages were seeded on smooth (pickled titanium (PT)) and rough Sand-blasted with Large grit particles followed by Acid-etching (SLA) titanium surfaces and first investigated for macrophage polarization towards tissue-inflammatory M1 macrophages or wound-healing M2 macrophages. Thereafter, culture media collected from macrophages on both surfaces were cultured with gingival fibroblasts seeded on their respective topographies. All experiments were performed in triplicate with three independent experiments. RESULTS: Macrophages seeded on SLA surfaces polarized towards tissue-inflammatory M1 macrophages at early time points. Immunofluorescent staining and RT-PCR analysis demonstrated higher levels of iNOS and gene expression of IL-1ß, IL-6, and TNF-alpha on SLA surfaces at 3 days when compared to both tissue culture plastic (TCP) and PT surfaces (p < 0.001). Very little differences were found between smooth PT surfaces and TCP. Interestingly, proliferation assay (CCK-8) suggested that conditioned media (CM) from macrophages seeded on SLA surfaces drastically inhibited gingival fibroblast proliferation at 3 and 5 days (p < 0.001). Meanwhile, CM from macrophages cultured on SLA surfaces also significantly reduced collagen 1 synthesis on SLA surfaces at 14 days as assessed by immunofluorescent staining (p < 0.001). CONCLUSION: The results from this study demonstrate that the polarization of macrophages towards a pro-inflammatory (M1) phenotype on SLA surfaces may have a negative impact on gingival fibroblast behavior on titanium surfaces. Future strategies to better modulate macrophage polarization should be investigated to support a favorable immune response and encourage tissue integration. CLINICAL RELEVANCE: As SLA surfaces have a potential to shift macrophages towards tissue-inflammatory M1 macrophages, this might be a negative impact for soft tissue healing. Therefore, SLA surfaces should be kept within the bone, as when in contact with soft tissue, they are prone to support a lack of soft tissue integration leading to inflammation.

Healing of two-wall intra-bony defects treated with a novel EMD-liquid-A pre-clinical study in monkeys.

AIM: To investigate the effect of a novel enamel matrix derivative formulation (EMD-liquid or Osteogain) combined with an absorbable collagen sponge (ACS) on periodontal wound healing in intra-bony defects in monkeys. MATERIALS AND METHODS: Chronic two-wall intra-bony defects were created at the distal aspect of eight teeth in three monkeys (Macaca fascicularis). The 24 defects were randomly assigned to one of the following treatments: (i) open flap debridement (OFD) + ACS alone, (ii) OFD + Emdogain + ACS (Emdogain/ACS), (iii) OFD + Osteogain + ACS (Osteogain/ACS) or (iv) OFD alone. At 4 months, the animals were euthanized for histologic evaluation. RESULTS: Osteogain/ACS resulted in more consistent formation of cementum, periodontal ligament and bone with limited epithelial proliferation compared to OFD alone, Emdogain/ACS and OFD + ACS. Among the four treatment groups, the Osteogain/ACS group demonstrated the highest amount of regenerated tissues. However, complete periodontal regeneration was not observed in any of the defects in the four groups. CONCLUSIONS: The present findings indicate that in two-wall intra-bony defects, reconstructive surgery with Osteogain/ACS appears to be a promising novel approach for facilitating periodontal wound healing/regeneration, thus warranting further clinical testing.

Variable expressivity of TCTEX1D2 mutations and a possible pathogenic link of molar-incisor malformation to ciliary dysfunction.

OBJECTIVE: Clarification of the molecular basis of a ciliopathy associated with molar-incisor malformation in a consanguineous Turkish family. DESIGN: Full dental and clinical examinations, histologic analysis, comprehensive genetic analyses including exome sequencing, ciliary function tests and transmission electron microscopy of ciliary biopsies in the surviving patient. RESULTS: Two siblings had situs inversus and complex heart defects suggestive of ciliary dysfunction. The affected girl who died in utero showed severe chest abnormalities compatible with Jeune syndrome which were not present in the affected boy. Dental investigations in the boy showed typical signs of molar-incisor-malformation. Exome sequencing identified a homozygous intragenic deletion in TCTEX1D2 which is predicted to completely remove protein function. Ciliary function tests and electron microscopy showed mild irregularities of motile cilia such as compound cilia and loss of membranes. CONCLUSIONS: Our findings support the suggestion that TCTEX1D2 mutations have variable expressivity and may be associated with disturbances of embryonic development caused by both, ciliary signaling and motile dysfunction. The presence of molar-incisor-malformation in the living patient raises the possibility of a pathogenetic link of this rare dental anomaly to ciliary dysfunction during tooth development at least in some individuals.

Combination of Collagen Barrier Membrane with Enamel Matrix Derivative-Liquid Improves Osteoblast Adhesion and Differentiation.

PURPOSE: Collagen barrier membranes were first introduced to regenerative periodontal and oral surgery to prevent fast ingrowing soft tissues (ie, epithelium and connective tissue) into the defect space. More recent attempts have aimed at combining collagen membranes with various biologics/growth factors to speed up the healing process and improve the quality of regenerated tissues. Recently, a new formulation of enamel matrix derivative in a liquid carrier system (Osteogain) has demonstrated improved physico-chemical properties for the adsorption of enamel matrix derivative to facilitate protein adsorption to biomaterials. The aim of this pioneering study was to investigate the use of enamel matrix derivative in a liquid carrier system in combination with collagen barrier membranes for its ability to promote osteoblast cell behavior in vitro. MATERIALS AND METHODS: Undifferentiated mouse ST2 stromal bone marrow cells were seeded onto porcine-derived collagen membranes alone (control) or porcine membranes + enamel matrix derivative in a liquid carrier system. Control and enamel matrix derivative-coated membranes were compared for cell recruitment and cell adhesion at 8 hours; cell proliferation at 1, 3, and 5 days; and real-time polymerase chain reaction (PCR) at 3 and 14 days for genes encoding Runx2, collagen1alpha2, alkaline phosphatase, and bone sialoprotein. Furthermore, alizarin red staining was used to investigate mineralization. RESULTS: A significant increase in cell adhesion was observed at 8 hours for barrier membranes coated with enamel matrix derivative in a liquid carrier system, whereas no significant difference could be observed for cell proliferation or cell recruitment. Enamel matrix derivative in a liquid carrier system significantly increased alkaline phosphatase mRNA levels 2.5-fold and collagen1alpha2 levels 1.7-fold at 3 days, as well as bone sialoprotein levels twofold at 14 days postseeding. Furthermore, collagen membranes coated with enamel matrix derivative in a liquid carrier system demonstrated a sixfold increase in alizarin red staining at 14 days when compared with collagen membrane alone. CONCLUSION: The combination of enamel matrix derivative in a liquid carrier system with a barrier membrane significantly increased cell attachment, differentiation, and mineralization of osteoblasts in vitro. Future animal testing is required to fully characterize the additional benefits of combining enamel matrix derivative in a liquid carrier system with a barrier membrane for guided bone or tissue regeneration.

Collagen barrier membranes adsorb growth factors liberated from autogenous bone chips.

OBJECTIVE: Collagen membranes serve as barriers to separate bone grafts from soft tissues. Bone grafts harvested with a bone scraper release growth factors activating transforming growth factor-ß (TGF-ß) signaling in mesenchymal cells. The aim of the present pilot study was to determine whether collagen membranes adsorb molecules from bone-conditioned medium (BCM) with the capacity to provoke the expression of TGF-ß target genes in vitro. MATERIALS AND METHODS: Collagen membranes were soaked in aqueous extracts from fresh and demineralized bone chips placed in cell culture medium. Recombinant human TGF-ß1 served as control. Gingival fibroblasts were seeded onto the washed collagen membranes and evaluated for the expression of adrenomedullin, pentraxin 3, interleukin 11, and proteoglycan 4. Cell viability and morphology with phalloidin staining were also determined. RESULTS: Incubation of collagen membranes with BCM for at least one minute caused fibroblasts to decrease the expression of adrenomedullin and pentraxin 3, and to increase the expression of interleukin 11 and proteoglycan 4. Four different membrane treatments - incubated with recombinant TGF-ß1, pre-wetted with saline solution, exposed to UV light, and dry out and stored one week at room temperature - also provoked significant changes in gene expression. Likewise, conditioned medium from demineralized bone chips caused gene expression changes. BCM did not alter the viability or morphology of gingival fibroblasts. CONCLUSIONS: These findings demonstrate that collagen membranes rapidly adsorb the TGF-ß activity released from bone chips, a molecular process that might contribute to guided bone regeneration.

Osteogain improves osteoblast adhesion, proliferation and differentiation on a bovine-derived natural bone mineral.

BACKGROUND: The use of enamel matrix derivative (EMD) has been shown to facilitate periodontal regeneration by histologically resulting in formation of cementum, periodontal ligament and bone. Recently, a new liquid carrier system for EMD has been introduced with better physicochemical properties specifically designed for bone graft mixing (Osteogain). The aim of this study was to investigate the combination of Osteogain with a bovine-derived natural bone mineral (NBM) on osteoblast migration, adhesion, proliferation and differentiation. MATERIALS AND METHODS: Undifferentiated mouse ST2 stromal bone marrow cells were seeded onto 1)NBM particles alone or 2)NBM + Osteogain. Samples were compared for cell migration at 8 h, cell adhesion at 4 h, cell proliferation at 1, 3 and 5 days and real-time PCR at 3 and 14 days for genes encoding runt-related transcription factor 2 (Runx2), collagen1alpha2 (COL1a2), alkaline phosphatase (ALP) and osteocalcin (OCN). Furthermore, alizarin red staining was utilized to investigate the mineralization at 14 days. RESULTS: Osteogain significantly upregulated cell adhesion over twofold onto NBM particles and promoted cell proliferation at 3 and 5 days after seeding. Furthermore, the combination of NBM with Osteogain significantly upregulated genes encoding Runx2, ALP, COL1a2 and OCN (from 1.5- to 3-fold) and increased alizarin red staining over 3 fold at 14 days when compared to NBM particles alone. CONCLUSION: Pre-coating Osteogain onto NBM bone grafting particles significantly increased cell adhesion, proliferation and differentiation of osteoblasts in vitro. Future animal studies are now necessary to further investigate the regenerative potential of Osteogain in combination with a bone grafting material prior to clinical use for bone regeneration.

Effects of EMD liquid (Osteogain) on periodontal healing in class III furcation defects in monkeys.

AIM: To evaluate the effect of a novel liquid carrier system of enamel matrix derivative (Osteogain) soaked on an absorbable collagen sponge (ACS) upon periodontal wound healing/regeneration in furcation defects in monkeys. MATERIALS AND METHODS: The stability of the conventional enamel matrix derivative (Emdogain) and Osteogain adsorbed onto ACS was evaluated by ELISA. Chronic class III furcation defects were created at teeth 36, 37, 46, 47 in three monkeys (Macaca fascicularis). The 12 defects were assigned to one of the following treatments: (1) open flap debridement (OFD) + ACS, (2) OFD+Emdogain/ACS, (3) OFD+Osteogain/ACS, and (4) OFD alone. At 16 weeks following reconstructive surgery, the animals were killed for histological evaluation. RESULTS: A 20-60% significantly higher amount of total adsorbed amelogenin was found for ACS-loaded Osteogain when compared to Emdogain. The histomorphometric analysis revealed that both approaches (OFD + Emdogain/ACS and OFD + Osteogain/ACS) resulted in higher amounts of connective tissue attachment and bone formation compared to treatment with OFD + ACS and OFD alone. Furthermore, OFD + Osteogain/ACS group showed higher new attachment formation, cementum, and new bone area. CONCLUSIONS: Within their limits, the present data indicate that Osteogain possesses favourable physicochemical properties facilitating adsorption of amelogenin on ACS and may additionally enhance periodontal wound healing/regeneration when compared to Emdogain.

The influence of collagen membrane and autogenous bone chips on bone augmentation in the anterior maxilla: a preclinical study.

OBJECTIVES: To evaluate the effect of a resorbable collagen membrane and autogenous bone chips combined with deproteinized bovine bone mineral (DBBM) on the healing of buccal dehiscence-type defects. MATERIAL AND METHODS: The second incisors and the first premolars were extracted in the maxilla of eight mongrels. Reduced diameter, bone-level implants were placed 5 weeks later. Standardized buccal dehiscence-type defects were created and grafted at implant surgery. According to an allocation algorithm, the graft composition of each of the four maxillary sites was DBBM + membrane (group D + M), autogenous bone chips + DBBM + membrane (group A + D + M), DBBM alone (group D) or autogenous bone chips + DBBM (group A + D). Four animals were sacrificed after 3 weeks of healing and four animals after 12 weeks. Histological and histomorphometric analyses were performed on oro-facial sections. RESULTS: The pattern of bone formation and resorption within the grafted area showed high variability among the same group and healing time. The histomorphometric analysis of the 3-week specimens showed a positive effect of autogenous bone chips on both implant osseointegration and bone formation into the grafted region (P < 0.05). The presence of the collagen membrane correlated with greater bone formation around the DBBM particles and greater bone formation in the grafted region after 12 weeks of healing (P < 0.05). The oro-facial width of the augmented region at the level of the implant shoulder was significantly reduced in cases where damage of the protection splints occurred in the first week of healing (P < 0.05). CONCLUSIONS: The addition of autogenous bone chips and the presence of the collagen membrane increased bone formation around DBBM particles. Wound protection from mechanical noxa during early healing may be critical for bone formation within the grafted area.

Giant cells around bone biomaterials: Osteoclasts or multi-nucleated giant cells?

Recently accumulating evidence has put into question the role of large multinucleated giant cells (MNGCs) around bone biomaterials. While cells derived from the monocyte/macrophage lineage are one of the first cell types in contact with implanted biomaterials, it was originally thought that specifically in bone tissues, all giant cells were bone-resorbing osteoclasts whereas foreign body giant cells (FBGCs) were found associated with a connective tissue foreign body reaction resulting in fibrous encapsulation and/or material rejection. Despite the great majority of bone grafting materials routinely found with large osteoclasts, a special subclass of bone biomaterials has more recently been found surrounded by large giant cells virtually incapable of resorbing bone grafts even years after their implantation. While original hypotheses believed that a 'foreign body reaction' may be taking place, histological data retrieved from human samples years after their implantation have put these original hypotheses into question by demonstrating better and more stable long-term bone volume around certain bone grafts. Exactly how or why this 'special' subclass of giant cells is capable of maintaining long-term bone volume, or methods to scientifically distinguish them from osteoclasts remains extremely poorly studied. The aim of this review article was to gather the current available literature on giant cell markers and differences in expression patterns between osteoclasts and MNGCs utilizing 19 specific markers including an array of CD-cell surface markers. Furthermore, the concept of now distinguishing between pro-inflammatory M1-MNGCs (previously referred to as FBGCs) as well as wound-healing M2-MNGCs is introduced and discussed. STATEMENT OF SIGNIFICANCE: This review article presents 19 specific cell-surface markers to distinguish between osteoclasts and MNGCs including an array of CD-cell surface markers. Furthermore, the concept of now distinguishing between pro-inflammatory M1-MNGCs (often previously referred to as FBGCs) as well as wound-healing M2-MNGCs is introduced and discussed. The proposed concepts and guidelines aims to guide the next wave of research facilitating the differentiation between osteoclast/MNGCs formation, as well as provides the basis for increasing our understanding of the exact function of MNGCs in bone tissue/biomaterial homeostasis.

High-dose irradiation of bone chips preserves the in vitro activity of bone-conditioned medium.

Extracorporeal irradiation sterilizes resected tumor bone used as autografts in reconstruction surgery. Therapeutic irradiation is a standard technique in head and neck cancer therapy that aims to preserve organ function. Bone irradiation has a complex, mostly inhibitory, effect on remodeling and regeneration, although the underlying mechanisms are still not fully understood. It remains unclear if extracorporeal irradiation affects the paracrine-like activity of the corresponding autografts. We recently reported that bone-conditioned medium from autogenous bone chips contains a number of factors that might affect cell activity. In the present study, we investigated the effects of extracorporeal irradiation of porcine cortical bone chips on the activity of the corresponding bone-conditioned medium. The effects of bone-conditioned medium on the expressions of transforming growth factor-beta (TGF-ß) target genes in oral fibroblasts were assessed. Bone-conditioned medium from bone chips exposed to a total radiation dose up to 120 Gy did not affect expressions of TGF-ß target genes, including adrenomedullin, BTB/POZ domain-containing protein 11, proteoglycan 4, NADPH oxidase 4, and interleukin 11, in oral fibroblasts. In conclusion, bone irradiation does not alter the capability of the corresponding bone-conditioned medium to provoke a robust fibroblastic cell response in vitro. (J Oral Sci 58, 325-331, 2016).