Isolation, characterization and investigation of differentiation potential of human periodontal ligament cells and dental follicle progenitor cells and their response to BMP-7 in vitro.

The aim of this study was to assess the factors, mechanisms and the differences between periodontal ligament (PDL) cells and denta l follicle (DF) progenitor cells towards the osteoblastic/cementoblastic differentiation and to investigate the effects of BMP-7 on developmental (DF) and mature tissue-derived (PDL) cells, respectively. Primary cell culture of PDL cells and DF progenitor cells was performed. Osteogenic differentiation was evaluated using von Kossa, Alizarin Red S and immuno-histo-chemistry staining of osteocalcin. Gene expression pattern was evaluated via real-time PCR. A series of CD surface marks were tested using flow cytometry and fluorescence-activated cell-sorting analysis was performed. Real-time RT-PCR demonstrated similar gene expression pattern of PDL cells and DF progenitor cells: the expression of OPN and OCN significantly was elevated when incubated with osteogenic components, Runx2 was unaffected, and Osteorix was hardly expressed whether in basic medium or induction medium. In addition, BMP-7 induced osteoblast/cementoblast differentiation of PDLSCs and DF progenitor cells in a dose- and time-dependent manner, as reflected by enhanced Runx2 and (OCN) mRNA transcript expression. BMP-7 triggers PDL cells and DF progenitor cells to differentiate towards an osteoblast/cementoblast phenotype.

Effect of deproteinized bovine bone matrix coverage on the resorption of iliac cortico-spongeous bone grafts - a prospective study of two cohorts.

OBJECTIVES: The augmentation of the alveolar ridge using iliac cortico-spongeous bone grafts is routinely used. However, bone grafts show a substantial degree of resorption, which may negatively affect the long-term success of dental implants in the augmented area. The aim of this study was to evaluate the effect of a deproteinized bovine bone matrix coverage on the resorption of iliac bone grafts. MATERIAL AND METHODS: Two cohorts consisting of 40 patients who received a vertical augmentation of the alveolar ridge with onlay grafts from the iliac crest were prospectively investigated over a period of 2 years. In half of the patients (n = 40), the grafts were covered by a thin layer of deproteinized bovine bone matrix (DBBM cohort). The other 40 patients received the identical surgical procedure without a DBBM coverage (non-DBBM cohort). The graft height/resorption was radiographically determined immediately after surgery, 6 months, 1 year, and 2 years postoperatively. RESULTS: The height of the bone graft 6 months after surgery accounted 92.15% of the initial value in the DBBM cohort and 87.76% in the non-DBBM cohort. One year after augmentation, the height reduced to 83.95% in the DBBM cohort and 72.92% in the non-DBBM cohort. Two years after surgery, the resorption slowed down and the height of the grafts accounted 81.27% in the DBBM cohort and 71.43% in the non-DBBM cohort. The difference was statistically significant. CONCLUSION: Deproteinized bovine bone matrix reduces the postoperative resorption of iliac bone block grafts and may therefore enhance the long-term implant prognosis in the augmented area.

Prevention of the surface resorption of bone grafts by topical application of bisphosphonate on different carrier materials.

OBJECTIVE: The hypothesis of the present study was that topically applied bisphosphonate (BP) on a collagen membrane or alternatively bovine bone mineral prevents surface resorption of onlay bone grafts. MATERIAL AND METHODS: In eight adult pigs, bone blocks were harvested bilaterally from the mandible and fixed to the lateral cortex of the horizontal ramus to simulate a ridge augmentation. In a split-mouth study design, we used alendronate in aqueous solution (1 mg/ml) on the test-side in three different ways: on a collagen membrane (Bio-Gide®), soaked in bovine bone mineral granules (Bio-Oss®), or applied to the bone graft directly. The same materials without BP were used as controls on the contralateral side. After 3 months, the animals were sacrificed. The evaluation included sequential fluorochromic labeling and measurement of bone height in microradiography and toluidine blue staining. RESULTS: In five cases, necrosis of the overlying periosteal tissues with BP was observed macroscopically. A statistically significantly lower loss in graft height was seen on the test-side for Bio-Gide® + alendronate (0.65 %) versus Bio-Gide® (1.52 %), p = 0.002; Bio-Oss® + alendronate (1.16 %) versus Bio-Oss® (4.20 %), p = 0.001; and bone graft + alendronate (1.25 %) versus bone graft alone (6.01 %), p = 0.006. An inhibitory effect on bone remodeling was observed by a statistically significantly lower number of resorption lacunae. CONCLUSION: The hypothesis was accepted that a bisphosphonate-treated membrane reduced bone graft resorption; however, periosteal necrosis requires better adaptation of the dosage. CLINICAL RELEVANCE: A bisphosphonate membrane could be a helpful tool to preserve augmentation height of onlay bone grafts.

Effects of zoledronate on irradiated bone in vivo: analysis of the collagen types I, V and their cross-links lysylpyridinoline, hydroxylysylpyridinoline and hydroxyproline.

Radiotherapy can lead to a reduction of bone density with an increased risk of pathological fractures. Bisphosphonates may represent a preventive treatment option by increasing the density of anorganic bone mineral. Yet it is unknown how bisphosphonates act on irradiated collagen cross-links, which play an essential role for the mechanical stability of bone. The aim of this study was to evaluate the effects of zoledronate on bone collagens and their cross-links after irradiation. The right femur of 37 rats was irradiated with a single dose of 9.5 Gy at a high dose rate using an afterloading machine. Half of the rats (n=18) received additionally a single dose zoledronate (0.1 mg/kg body weight). Fourteen and 100 days after irradiation the femora were collected for histologic evaluation and determination of the collagen cross-links lysylpyridinoline, hydroxylysylpyridinoline, and hydroxyproline. The collagen types were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Fourteen days after treatment the lysylpyridinoline levels of all treatment groups were significantly lower compared to the untreated control. After 100 days, in the combined radiotherapy+zoledronate group significantly lower lysylpyridinoline values were determined (p=0.009). Radiotherapy and/or zoledronate did not change significantly the level of hydroxylysylpyridinoline. The concentration of hydroxyproline was 14 days after irradiation significantly higher in the combined treatment group compared to the control. No significant differences were observed 100 days after treatment. Zoledronate does not have the ability to restore the physiological bone collagen cross-link levels after radiotherapy. However, this would be necessary for regaining the physiological mechanical stability of bone after irradiation and therefore to prevent effectively radiation-induced fractures.

Orbital floor reconstruction with resorbable polydioxanone implants.

Many different materials are proposed for reconstruction of traumatic orbital floor defects. Donor-site morbidity of autologous transplants and infections or extrusions of nonresorbable implants lead to a widespread use of resorbable, alloplastic materials such as polydioxanone (PDS). The goal of this study was to evaluate the prevalence of orbital floor fracture-related problems after surgical treatment using PDS. Ophthalmologic and clinical examinations were performed at 194 patients before orbital floor reconstruction, 14 days and 6 months after surgery (approximate defect sizes: <1 cm², n=50; 1-2 cm², n=97; >2 cm², n=47). Clinical findings including the ocular motility, the sensibility of the infraorbital nerve, and the position of the globe were evaluated. For statistical analysis of categorical data, confidence intervals of percentages were determined. Linear relationships between 2 variables were assessed with Pearson correlation analysis. A reduced ocular motility was diagnosed in 60 patients (31%) before surgery; in 14 patients (7%), 2 weeks; and in 10 patients (5%), 6 months after surgery. Infraorbital hypesthesia was found in 120 patients (62%) before surgery; in 47 patients (24%), 2 weeks; and in 35 patients (18%), 6 months after surgery. An enophthalmos was present in 10 patients (5%) before surgery, and in 4 patients (2%), 6 months after surgery. Our data suggest that PDS is a suitable implant for orbital floor reconstruction with acceptable low rates of infraorbital hypesthesia, bulbus motility disturbances, and enophthalmos. Polydioxanone can also be used for orbital floor defects exceeding 2 cm².

Suppression of osteoblast-related genes during osteogenic differentiation of adipose tissue derived stromal cells.

Recent studies indicated a lower osteogenic differentiation potential of adipose tissue-derived stromal cells (ASCs) compared to bone marrow derived mesenchymal stromal cells. The aim of this study was to evaluate the effects of potent combinations of highly osteogenic bone morphogenetic proteins (BMPs) in order to enhance the osteogenic differentiation potential of ASCs. Human ASCs were cultured for 10 days in the presence of osteogenic medium consisting of dexamethasone, ß-glycerophosphate and ascorbat-2-phosphate (OM) supplemented with BMP-2, BMP-6, BMP-9+IGF-2 and BMP-2,-6,-9 (day 1+2: 50 ng/ml, days 3-6: 100 ng/ml, days 7-10: 200 ng/ml). The formation of the osteoblast phenotype was evaluated by quantification of osteoblast-related marker genes using real-time polymerase chain reaction (RT-PCR). Matrix mineralization was assessed by Alizarin Red S staining. Statistical analysis was carried out using the one-way analysis of variance (ANOVA) followed by the Scheffe's post hoc procedure. Osteogenic medium (OM) significantly increased the expression of alkaline phosphatase (ALP) and osteocalcin (p < 0.05) and led to a stable matrix mineralization. Under the influence of BMP-9+IGF-2 and BMP-2,-6,-9 the ALP expression further increased compared to ASCs cultured with OM only (p < 0.01). However, multiple osteogenic markers showed no change or decreased under the influence of OM and BMP combinations (p < 0.05). The current results indicate a restricted osteogenic differentiation potential of ASCs and suggest careful reconsideration of their use in bone tissue engineering applications.

In Vitro Assessment of Primary Stability of BoneTrust Sinus Implant Design.

The aim of this study was to analyze the primary stability of BoneTrust Sinus implants (BTSIs), which are intended to enable higher primary stability by their special design with reduced thread section in cases of reduced vertical bone availability, in comparison with standard BoneTrust implants (SBTIs) in vitro. A bone window 3 cm in length, 4 cm in width, and 3 cm in depth, resembling the maxillary bone window of the lateral sinus wall with 4 mm of residual bone height, was prepared at the dorsal side of freshly slaughtered bovine ribs. One single BTSI and a single SBTI with the same diameter (4 or 5 mm) were placed in each window. After implant placement, the implant stability quotient (ISQ) was measured by using resonance frequency analysis with an Osstell device. A total of 88 implants were placed. ISQ values varied between 63 and 84. Among the implants with 4-mm diameter, all BTSIs showed higher ISQ values compared with SBTIs. One-way analysis of variance showed a significant difference between BTSIs/SBTIs (P < .05). BTSIs with 4-mm diameter showed statistically higher values compared to BTSIs with 5-mm diameter (P < .05). Among the implants with 5-mm diameter, all SBTIs showed higher ISQ values compared to BTSIs but there was no significant difference. The use of 4-mm-diameter BTSIs could present higher ISQ values during simultaneous implant placement in conjunction with lateral sinus floor augmentation.

Improved adipogenic in vitro differentiation: comparison of different adipogenic cell culture media on human fat and bone stroma cells for fat tissue engineering.

To date there is no sufficient in vitro fat tissue engineering and a protocol has not been well established for this purpose. Therefore, we evaluated the in vitro influence of two different adipogenic growth media for their stimulation potential on different cell lineages to clearly define the most potent adipogenic growth media for future in vitro tissue engineering approaches. The samples for differentiation were composed of human adipogenic-derived stroma cells (hADSCs) and human bone marrow mesenchymal stroma cells (hMSCs). A normal adipogenic medium (NAM) and a specific adipogenic medium (SAM) were tested for their adipogenic stimulation potential. After 10 days and 21 days the relative gene expression was measured for the adipogenic marker genes PPARγ2, C/EBPα, FABP4, LPL, and GLUT4 detected through real time reverse transcriptase polymease chain reaction (RT-PCR). Other study variables were the comparison between NAM and SAM and between the used cells hADSCs and hMSCs. Additionally an Oil-Red staining was performed after 21 days. Our results revealed that only SAM was significantly (P<0.05) superior in the differentiation process in contrast to NAM for 10 days and 21 days. As well was SAM superior to differentiate the used cell lineages. This was evaluated by the detected marker genes PPARγ2, C/EBPα, FABP4, LPL, and GLUT4 through real time RT-PCR and by Oil-Red staining. In addition, the hMSCs proofed to be equal donor cells for adipogenic differentiation especially when stimulated by SAM. The results suggest that the SAM should be established as a new standard medium for a more promising in vitro adipogenic differentiation.

Distraction histogenesis of the maxillofacial region.

The changes in the surrounding soft tissues during long bone distraction in orthopedic surgery have been the subject of several reports, studies on changes in the craniofacial region, in which various tissues, including the skin, muscle, tendon, blood vessel, and gingiva are rare. Therefore, there is a need for studies on the soft tissue aspects of bone lengthening of the craniofacial region. The aim of this review was to address this issue by reviewing the literature about the distraction histogenesis of various tissues, including skin, muscle, blood vessel, nerve, and gingiva.

Collection of Wound Exudate From Human Digit Tip Amputations Does Not Impair Regenerative Healing: A Randomized Trial.

The regrowth of amputated digit tips represents a unique regenerative healing in mammals with subcutaneous volume regrowth, restoration of dactylogram, and suppression of scar formation. Although factor analysis in amphibians and even in mice is easy to obtain, safety of harvesting biomaterial from human digit tip amputations for analysis has not yet been described.The aim of this study was to evaluate if recovering wound exudate does hamper clinical outcome or influence microbiologic or inflammation status.A predefined cohort of 18 patients with fresh digit tip amputations was randomly assigned to receive standard therapy (debridement, occlusive dressing) with (n = 9) or without (n = 9) collection of the whole wound exudate in every dressing change. Primary endpoint (lengthening) and secondary endpoints (regeneration of dactylogram, nail bed and bone healing, time to complete wound closure, scar formation, 2-point discrimination, microbiologic analysis, inflammatory factors interleukin (IL)-1α, tumor necrosis factor-α, IL-4, and IL-6) were determined by an independent, blinded observer.Patients' characteristics showed no significant differences between the groups. All patients completed the study to the end of 3 months follow-up. Exudate collection did not influence primary and secondary endpoints. Furthermore, positive microbiologic findings as well as pus- and necrosis-like appearance neither impaired tissue restoration nor influenced inflammatory factor release.Here, the authors developed an easy and safe protocol for harvesting wound exudate from human digit tip amputations. For the first time, it was shown that harvesting does not impair regenerative healing. Using this method, further studies can be conducted to analyze regeneration associated factors in the human digit tip.DRKS.de Identifier: DRKS00006882 (UTN: U1111-1166-5723).

Optimizing the osteogenic differentiation of human mesenchymal stromal cells by the synergistic action of growth factors.

A variety of different growth factors, most notably bone morphogenetic proteins (BMPs), have been shown to stimulate the osteogenic differentiation of mesenchymal stromal cells (MSCs) in vitro. Yet, due to the lack of comparative studies it remains unclear which protocol is the most effective in the induction of osteogenesis in MSC cultures. The aim of this study was to compare the most potent growth factors in regard to their osteoinductive potential. Human MSCs were cultured for 10 days in the presence of BMP-2, BMP-6, BMP-9 + IGF-2 and BMP-2, -6, -9 (day 1 + 2: 50 ng/ml; days 3-6: 100 ng/ml; days 7-10: 200 ng/ml). The formation of the osteoblast phenotype was assessed by quantification of osteoblast-related marker genes using reverse transcription polymerase chain reaction (RT-PCR) and alkaline phosphatase (ALP) staining. Matrix mineralization was assessed by alizarin red S and von Kossa staining. Statistical analysis was carried out using the one-way analysis of variance (ANOVA) followed by Scheffe's post hoc procedure. Among the tested growth factors the combination of BMP-2 + BMP-6 + BMP-9 most effectively induced the upregulation of collagen type I, collagen type V, osteocalcin, alkaline phosphatase, RUNX2, BMP-2, osteonectin and DLX5 (p < 0.01) and resulted in a consistent matrix mineralization. The findings suggest the combined addition of BMP-2, BMP-6 and BMP-9 to the osteoinductive culture medium containing dexamethasone, ß-glycerophosphate and ascorbate-2-phosphate produces more potent osteoblast differentiation of human MSCs in vitro.

Assessment of the lateral pterygoid plate fractures during Le Fort I osteotomies regarding the angulation of the osteotome.

AIM. This study aimed to clarify the relation between the angulation of the curved osteotome and fracture of the pterygoid plate during Le Fort I osteotomy. MATERIAL AND METHODS. Twenty-one specimens of hemisectioned Turkish skulls were used for the study. The maxilla was sectioned transversely on the floor of the pyriform aperture and posteriorly to the lateral pterygoid plate with a mechanical saw. The pterygomaxillary junction was separated with a curved osteotome by angulating the osteotome with, 0° and -30° to the occlusal plane. The undesired fractures of the lateral pterygoid plate were determined. Among 21 specimens, 7 pterygomaxillary junctions were separated with an angle of +30° , 7 with 0° and 7 with -30° to the occlusal plane. RESULTS. In group +30°, the undesired fracture occured in 6 of the cases. In group -30°, the undesired fracture was determines in one case. In cases where the separation was performed by placing the osteotome paralell to the occlusal plane all plates remained safe. CONCLUSION. Within the limited knowledge of the current study it can be concluded that the osteotome should be placed paralell to the occlusal plane.

Effects of different scaffolds on rat adipose tissue derived stroma cells.

BACKGROUND: Adipose tissue derived stroma cells (ASC's) offer for many advantages for tissue engineering strategies over mesenchymal stroma cells from other sources and ideal carrier materials have to be identified for them. The aim of this study was to demonstrate and to compare the effects of three clinically established biomaterials on proliferation and metabolic activity of rat ASC's in vitro. MATERIALS AND METHODS: Rat adipose tissue derived stroma cells (ASC's) were isolated and differentiated into distinct lineages proved by lineage specific staining and gene expression analysis (RT-PCR). The biomaterials Bio-Gide(®), Tutodent(®) Membrane and Belotero(®) Soft were tested with rat ASC's for their biocompatibility using scanning electron microscopy (SEM), cell vitality staining, cytotoxicity and proliferation tests (LDH, MTT, BrdU, WST-1). RESULTS: The collagen membrane Bio-Gide(®) resulted in a significantly higher viability and proliferation (WST-1, BrdU) compared to Tutodent(®) Membrane. No significant difference was determined in the LDH and MTT test. The hyaluronic acid gel Belotero(®) Soft showed no cytotoxicity (LDH, FDA/PI) and had no negative effects on metabolic activity (WST-1, MTT) or cell proliferation (BrdU) of ASC's. CONCLUSION: Our results indicate Bio-Gide(®) and Belotero(®) Soft as preferable carrier materials for ASC's. For the further establishment of ASC's-based treatment strategies, in vivo investigations on the tissue regeneration potential of these cell-biomaterial scaffolds should follow.

Comparison of two different absorbable membranes for the coverage of lateral osteotomy sites in maxillary sinus augmentation: a preliminary study.

INTRODUCTION: Barrier membranes, both absorbable and non-absorbable, have been used in sinus augmentation for many years. Some years ago, a new autologous blood substrate called Platelet-Rich-Fibrin (PRF) was introduced, and to date, the supporting effect on bone regeneration has been controversial. This study aimed to evaluate the effect of PRF on bone regeneration when used as a barrier membrane at the lateral osteotomy site in sinus augmentation. MATERIAL AND METHODS: Twelve sinuses from six patients requiring bilateral sinus floor augmentation were treated with a two-stage surgical technique using sinus augmentation and implant placement after 5 months. The sinuses were grafted with autologous bone and bone-substitute material (Bio-Oss(®)) mixed in a 1:1 ratio and were covered in a randomized split-mouth design with a PRF or a conventional collagen membrane (Bio-Gide(®)), respectively. Five months later threaded titanium dental implants were inserted and bone specimens harvested with a trephine burr were evaluated histomorphometrically. RESULTS: Bone quality seemed to be equal at both sites of the grafted sinuses. Mean vital bone formation after 5 months was 17.0% and 17.2%, for the PRF and collagen sites, respectively. The mean of residual bone-substitute was 15.9% and 17.3% for PRF and collagen, respectively. No local complications, such as dehiscences or membrane exposures, were detected at either site in any of the treated patients. After 12 months all implants reached primary stability in the augmented maxillary sinus floor without any peri-implant tissue inflammation. CONCLUSIONS: Within the limits of the study the coverage of the lateral sinus window with two different absorbable membranes has been shown to result in a similar amount of vital bone formation and residual bone-substitute.

Aging changes of the midfacial fat compartments: a computed tomographic study.

BACKGROUND: The restoration of a natural volume distribution is a major goal in facial rejuvenation. The aims of this study were to establish a radiographic method enabling effective measurements of the midfacial fat compartments and to compare the anatomy between human cadavers of younger versus older age. METHODS: Data from computed tomographic scans of 12 nonfixed cadaver heads, divided into two age groups (group 1, 54 to 75 years, n = 6; and group 2, 75 to 104 years, n = 6), were analyzed. For evaluation of the volume distribution within a specific compartment, the sagittal diameter of the upper, middle, and lower thirds of each compartment was determined. For evaluation of a "sagging" of the compartments, the distance between the cephalad border and the infraorbital rim was determined. RESULTS: Computed tomography enables a reproducible depiction of the facial fat compartments and reveals aging changes. The distance between the fat compartments and the infraorbital rim was higher in group 2 compared with group 1. The sagittal diameter of the lower third of the compartments was higher, and the sagittal diameter of the upper third was smaller in group 2 compared with group 1. The buccal extension of the buccal fat pad was shown to be an independent, separate compartment. CONCLUSIONS: This study demonstrates an inferior migration of the midfacial fat compartments and an inferior volume shift within the compartments during aging. Additional distinct compartment-specific changes (e.g., volume loss of the deep medial cheek fat and buccal extension of the buccal fat pad) contribute to the appearance of the aged face.

Survival of transplanted rat bone marrow-derived osteogenic stem cells in vivo.

This study was designed to trace bone marrow-derived stromal cells (MSC) after implantation in an ectopic rat model of bone tissue engineering. MSC were isolated from adult donor rats, expanded, seeded on a hydroxyapatite/ß-tricalcium phosphate bone graft substitute (Straumann® BoneCeramic), and cultivated until confluent. Before subcutaneous implantation of seeded constructs and controls (unseeded bone graft substitute) in isogenic rats (n = 32), cells were labeled with the fluorescent dye carboxyfluoresceine-diacetate-succinimidyl-ester. Specimens were harvested at sacrifice on day 1, 3, 7, or 14 after implantation (n = 8 per group) and processed for histology (hematoxylin and eosin, CD68, 4',6-diamidino-2-phenylindol). Carboxyfluoresceine-diacetate-succinimidyl-ester-labeled transplanted cells were quantified in decalcified sections (50 fields of view per specimen) at 488 nm. Over time, transplanted cells decreased in number from 31.3 ± 2.3 (day 1) to 9.2 ± 1.1 (day 3) and 0.3 ± 0.1 (day 7) (p < 0.001). Fourteen days postimplantation MSC could no longer be identified. Additionally, starting on day 3 postimplantation, cellular disintegration was noted. Multinucleated giant cells were present in constructs and controls on day 7 and increased to day 14 postimplantation. These results indicate that ectopically transplanted MSC survive for a rather short time after implantation. Possible reasons for early cell death are discussed.