Tissue-engineered bone construct promotes early osseointegration of implants with low primary stability in oversized osteotomy.

OBJECTIVES: To evaluate the histological parameters and bone mechanical properties around implants with low primary stability (PS) in grafted bone substitutes within an oversized osteotomy. MATERIALS AND METHODS: An oversized osteotomy penetrating the double cortical bone layers was made on both femora of 24 New Zealand white rabbits. Bilaterally in the femur of all animals, 48 implants were installed, subdivided into four groups, corresponding to four prepared tissue-engineering bone complexes (TEBCs), which were placed between the implant surface and native bone wall: A: tricalcium phosphate ß (TCP-ß); B: autologous adipose derived-stem cells with TCP-ß (ASCs/TCP-ß); C: ASCs transfected with the enhanced-GFP gene with TCP-ß (EGFP-ASCs/TCP-ß); D: ASCs transfected with the BMP-2 gene with TCP-ß (BMP2-ASCs/TCP-ß). Trichrome fluorescent labeling was conducted. Animals were sacrificed after eight weeks. The trichromatic fluorescent labeling (%TFL), area of new bone (%NB), residual material (%RM), bone-implant contact (%BIC), and the removal torque force (RTF, N/cm) were assessed. RESULTS: ASCs were successfully isolated from adipose tissue, and the primary ASCs were induced into osteogenic, chondrogenic, and adipogenic differentiation. The BMP-2 overexpression of ASCs sustained for ten days and greatly enhanced the expression of osteopontin (OPN). At eight weeks post-implantation, increased %NB and RTF were found in all groups. The most significant value of %TFL, %BIC and lowest %RM was detected in group D. CONCLUSION: The low PS implants osseointegrate with considerable new bone in grafted TEBCs within an oversized osteotomy. Applying BMP-2 overexpressing ASCs-based TEBC promoted earlier osseointegration and more solid bone mechanical properties on low PS implants. Bone graft offers a wedging effect for the implant with low PS at placement and promotes osteogenesis on their surface in the healing period.

Local delivery of simvastatin maintains tooth anchorage during mechanical tooth moving via anti-inflammation property and AMPK/MAPK/NF-kB inhibition.

Simvastatin (SMV) could increase tooth anchorage during orthodontic tooth movement (OTM). However, previous studies on its bone-specific anabolic and anti-inflammation properties were based on static in vitro and in vivo conditions. AMPK is a stress-activated kinase that protects tissue against serious damage from overloading inflammation. Rat periodontal ligament cells (PDLCs) were subjected to a serial of SMV concentrations to investigate the optimization that promoted osteogenic differentiation. The PDLCs in static and/or tensile culturing conditions then received the proper concentration SMV. Related factors expression was measured by the protein array, real-time PCR and Western blot. The 0.05UM SMV triggered osteogenic differentiation of PDLCs. The inhibition of AMPK activation through a pharmacological approach (Compound C) caused dramatic decrease in osteogenic/angiogenic gene expression and significant increase in inflammatory NF-κB phosphorylation. In contrast, pharmacological activation of AMPK by AICAR significantly inhibited inflammatory factors expression and activated ERK1/2, P38 MAPK phosphorylation. Moreover, AMPK activation induced by SMV delivery significantly attenuated the osteoclastogenesis and decreased the expression of pro-inflammatory TNF-α and NF-κB in a rodent model of OTM. The current studies suggested that SMV could intrigue intrinsic activation of AMPK in PDLCs that promote attenuate the inflammation which occurred under tensile irritation through AMPK/MAPK/NF-kB Inhibition.

Hybrid system with stable structure of hard/soft tissue substitutes induces re-osseointegration in a rat model of biofilm-mediated peri-implantitis.

Re-osseointegration of an infected/contaminated dental implant poses major clinical challenges. We tested the hypothesis that the application of an antibiotic-releasing construct, combined with hard/soft tissue replacement, increases the efficacy of reconstructive therapy. We initially fabricated semi-flexible hybrid constructs of ß-TCP/PHBHHx, with tetracycline (TC) (TC amounts: 5%, 10%, and 15%). Thereafter, using in vitro assays, TC release profile, attachment to rat bone marrow-derived stem cells (rBMSCs) and their viability as well as anti-bacterial activity were determined. Thereafter, regenerative efficacies of the three hybrid constructs were assessed in a rat model of peri-implantitis induced by Aggregatibacter actinomycetemcomitans biofilm; control animals received ß-TCP/Bio-Gide and TC injection. Eight weeks later, maxillae were obtained for radiological, histological, and histomorphometric analyses of peri-implant tissues. Sulcus bleeding index was chronologically recorded. Serum cytokines levels of IL-6 and IL-1ß were also evaluated by enzyme-linked immunosorbent assay. Substantial amounts of tetracycline, from hybrid constructs, were released for 2 weeks. The medium containing the released tetracycline did not affect the adhesion or viability of rBMSCs; however, it inhibited the proliferation of A. actinomycetemcomitans. Osteogenesis and osseointegration were more marked for the 15% hybrid construct group than the other two groups. The height of attachment and infiltration of inflammatory cells within fibrous tissue was significantly reduced in the experimental groups than the control group. Our protocol resulted in re-osseointegration on a biofilm-contaminated implant. Thus, an antibiotic releasing inorganic/organic construct may offer a therapeutic option to suppress infection and promote guided tissue regeneration thereby serving as an integrated multi-layer substitute for both hard/soft tissues.

Maxillary sinus floor elevation using BMP-2 and Nell-1 gene-modified bone marrow stromal cells and TCP in rabbits.

This study evaluated the synergistic osteogenic effect of bone morphogenetic protein-2 (BMP-2) and Nel-like molecule-1 (Nell-1) genes in a rabbit maxillary sinus floor elevation model. Bone marrow stromal cells (bMSCs) were cultured and transduced with AdEGFP, AdNell-1, AdBMP-2, or AdNell-1 + AdBMP-2 overexpression virus. These gene-modified autologous bMSCs were then combined with a ß-tricalcium phosphate (ß-TCP) granule scaffold and used to elevate the maxillary sinus floor in rabbits. bMSCs cotransduced with AdNell-1 + AdBMP-2 demonstrated a synergistic effect on osteogenic differentiation as detected by real-time PCR analysis on markers of runt-related transcription factor-2, osteocalcin, collagen type 1, alkaline phosphatase activity, and calcium deposits in vitro. As for maxillary sinus floor elevation in a rabbit model in vivo, AdNell-1 + AdBMP-2 gene-transduced autologeous bMSCs/ß-TCP complex had the largest bone area and most mature bone structure among the groups, as detected by HE staining and immunohistochemistry at weeks 2 and 8 after implantation. Our data suggested that the BMP-2 and Nell-1 genes possessed a synergistic effect on osteogenic differentiation of bMSCs, while bMSCs modified with the BMP-2 and Nell-1 genes could promote new bone formation and maturation in the rabbit maxillary sinus model.

[Research of TC-PHBHHx/ß-TCP compounds for treatment of dry socket in SD rats].

PURPOSE: The effects of different compounds on dry socket were evaluated in order to find a new method that can both be antibacterial and osteogenic,providing experimental evidence for future clinical application. METHODS: Seventy-two male SD rats, with upper left anterior teeth been extracted, were infected by pus to result in dry socket.Seven days later, they were allocated randomly and evenly into 4 groups and received different treatment, i.e. group A: debridement; group B: debridement and filled with iodoform gauzes; group C: debridement and filled with periocline; group D: debridement and filled with TC-PHBHHx/ß-TCP. After being treated for 1,4,8 weeks, sequential fluorescent labeling was performed. The animals were sacrificed after the procedure and hard tissue and decalcified sections were harvested for histological and histomorphometrical evaluation. Statistical analysis was performed using SPSS 19.0 software package. RESULTS: At the same time point, the results of osteogenesis in group A, B and C were not significantly different while the results in group D was significantly different from other groups; accordingly, significant new bone formation was observed. At different observation time, the bone area in group B and C were not significant different over time. In group A, little new bone formation was found but surrounded by a large amount of inflammatory cells at 8 week. However, group D showed bone area increasing gradually with time. CONCLUSIONS: TC-PHBHHx/ß-TCP has significant anti-inflammatory and osteogenic effects. Iodoform gauzes and periocline have good anti-inflammatory results but not significant osteogenic effects.

[Expression and significance of secreted frizzled-related protein 1 and ß-catenin in gingival tissue of patients with chronic periodontitis].

OBJECTIVE: This study aimed to investigate the expression and correlation of secreted frizzled-related protein 1 (SFRP1) and ß-catenin in gingival tissues of patients with chronic periodontitis (CP). The role of the classical Wnt/ß-catenin signaling pathway in the development of periodontitis was also explored. METHODS: Twenty-eight patients with CP (CP group) were enrolled in this study. Among them, 16 cases were moderate CP, and 12 demonstrated severe CP. Twelve healthy cases comprised the controls (normal group). Gingival tissue was collected, and the probing depth, bleeding index, and clinical attachment loss were recorded. The expression levels of SFRP1 and ß-catenin were detected by immunohistochemistry, and staining intensity was evaluated by double scoring method. SPSS 19.0 was used for statistical analysis. RESULTS: The staining strength scores of SFRP1 and ß-catenin were 2.16±0.65 and 1.12±0.51 in the normal group, 3.57±0.45 and 2.36±0.49 in the CP group, 3.61±0.40 and 2.30±0.44 in the moderate CP group, and 3.52±0.52 and 2.45±0.55 in the severe CP group, respectively. The expression of SFRP1 and ß-catenin in the CP group was higher than that in the normal group (P<0.01). A significant difference was noted between the normal group and the moderate and severe CP groups (P<0.01) but none between the moderate and severe CP groups (P>0.05). A positive correlation was found between the expression of SFRP1 and ß-catenin (r=0.657, P<0.01). The expression levels of ß-catenin and SFRP1 were related to periodontal indexes. The correlation between the expression of SFRP1 and probing depth was most significant (r=0.723, P<0.01), as well as that between ß-catenin and bleeding index (r=0.697, P<0.01). CONCLUSIONS: Patients with CP exhibit elevated expression of SFRP1 and ß-catenin in gingival tissues, and this event is related to the degree of periodontal destruction. Abnormal expression of SFRP1 and ß-catenin may promote the development of periodontitis.

[Detection of Dickkopf-1 and alkaline phosphatase activity in gingival crevicular fluid from chronic periodontitis with Er:YAG laser as an adjunctive treatment].

PURPOSE: The aim of this study was to evaluate the clinical efficacy and the level of DKK1 and alkaline phosphatase (ALP) activity in gingival crevicular fluid (GCF) while taking Er:YAG laser as an adjunctive to scaling and root planning in the treatment of chronic periodontitis (CP). METHODS: Eleven patients with CP were included and there were nineteen pairs of homonym teeth(thirty-eight teeth) in this split-mouth design, and they were randomly assigned to experimental group or control group. In the experimental group, a combination of ultrasonic subgingigval scaling and root planning with hand instrument (SRP) were performed with Er: YAG laser as an adjunctive; in the control group, only SRP was performed. The main variables were bleeding index (BI), probing depth (PD), clinical attachment loss (CAL) which were assessed at baseline (1 week after ultrasonic subgingival scaling), l month and 3 months after treatment. GCF was collected at baseline, l week, l month and 3 months, and the levels of DKK1 and ALP activity were detected at the same time point. The data were analyzed with SPSS 19.0 software package. RESULTS: Both groups showed significant reduction of PD, CAL, BI values 1 month and 3months after treatment, but no significant difference in clinical parameters were found between the two groups. In the experimental group, the activity of ALP reduced to (386.69±146.42), (341.221±171.62), (249.27±98.72) from (396.191±150.55) U/L and the level of DKK1 dropped to (310.34±184.68), (270.04±55.14), (247.31±56.99) from (307.12±45.63) µg/L at the end of 1 week, 1 month, 3 months, respectively. Meanwhile, in the control group, the activity of ALP reduced to (374.72±131.27), (344.42±127.80), (252.36±90.4 ) from (394.09±120.25) U/L and the level of DKK1 dropped to (310.34±84.68), (270.04±55.14), (247.31±56.99) from (305.33±147.40) µg/L at the end of l week, l month, 3months, respectively. There is no significant difference between the two groups at any period for ALP or DKK1. CONCLUSIONS: Er:YAG laser was a safe no-surgical adjunctive therapy in treating chronic periodontitis, further observation is needed to determine its long-term effectiveness.

Reosseointegration Following Regenerative Therapy of Tissue-Engineered Bone in a Canine Model of Experimental Peri-Implantitis.

BACKGROUND: Due to the existence of inflammation and limited osteogenesis on the precontaminated implant surface, reosseointegration is difficult to realize by current therapies. Tissue-engineering strategy has been proved quite effective in intractable bone defect situation. PURPOSE: This study was designed to see whether the adoption of tissue-engineered bone complex of adipose-derived stem cells (ASCs) and bone morphogenetic protein-2 (BMP-2) gene delivery would work efficiently in the correction of experimental peri-implantitis. METHODS: All premolars in both side of mandibular were removed from six beagle canines three months before implant placement. Typical peri-implantitis were then induced by three month ligature placement. After the implementation of identical anti-bacterial and mechanical debridement therapy, the shaped peri-implant defect were stuffed with four groups of constructs, as A: beta tricalcium phosphate (ß-TCP); B: ß-TCP with ASCs; C: ß-TCP with enhanced green fluorescent protein gene transduced ASCs (AdGFP-ASCs); and D: ß-TCP with bone morphogenetic protein-2 gene-modified ASCs (AdBMP-2-ASCs). Systematic radiographic, micro-CT, and histomorphometrical assessments were performed. RESULTS: After six months of healing, more bone formation and reosseointegration was found around the implant of groups B and C than group A. And group D further promoted the new bone height and reosseointegration percentage. Moreover, sequential fluorescence labeling tells that group D exhibited the quickest and strongest bone formation on the cleaned implant surface during the entire observation period as compared to the other three groups. CONCLUSIONS: These data demonstrated that tissue engineered bone of ASCs, BMP-2 gene delivery, and ß-TCP could exert powerful therapeutic effect on peri-implantitis as expected, which may suggest a feasible way to maintain the stability and masticatory function of dental implant.

Comprehensive Evaluation of Cryopreserved Bone-Derived Osteoblasts for the Repair of Segmental Mandibular Defects in Canines.

BACKGROUND: The repair of segmental mandibular defects remains challenging in the clinic. Previous studies have shown that cryopreserved bone-derived osteoblasts (CBOs) have good proliferation and osteogenicity. However, whether these cells can be used in the repair of segmental mandibular defects is largely unknown. PURPOSE: In this study, we applied CBOs combined with beta-tricalcium phosphate (ß-TCP) to repair a segmental mandibular defect in canines and thus established the feasibility of using this type of tissue-bank cell for the repair of large bone defects in the future. MATERIAL AND METHODS: Sixteen segmental mandibular defects in 16 animals were made on the right side. Sequential radiographs, computer tomography, polychrome fluorescent labeling, immunohistochemical staining, and histological analysis were used to evaluate the effects of tissue-engineered bone for segmental mandibular defects. RESULTS: Our results demonstrated that CBOs combined with ß-TCP promoted bone mineralization and deposition at the early stage, and bony union was achieved in the CBO and fresh bone-derived osteoblast (FBO) groups. However, nonunion and minimal callus were present in the ß-TCP group. Furthermore, there was a large amount of newly formed bone in the CBO and FBO groups and in the autogenous bone group. Additionally, osteocalcin immunohistochemistry showed intensive osteocalcin immunoreactivity in the bone matrix of the CBO and FBO groups. CONCLUSIONS: These data indicate that CBOs implanted in a scaffold can promote new bone formation, and this tissue-engineered bone can repair critically sized segmental mandibular defects in canines. The use of CBOs combined with ß-TCP may be an effective approach for the reconstruction of segmental mandibular defects in the clinic.

Maintenance of phenotype and function of cryopreserved bone-derived cells.

The emerging fields of tissue engineering and regenerative medicine require large numbers of cells for therapy. Although the properties of cells obtained from a variety of fresh tissues have been delineated, the knowledge regarding cryopreserved grafts-derived cells remains elusive. Previous studies have shown that living cells could be isolated from cryopreserved bone grafts. However, whether cryopreserved bone-derived cells can be applied in regenerative medicine is largely unknown. The present study was to evaluate the potential application of cryopreserved grafts-derived cells for tissue regeneration. We showed that cells derived from cryopreserved bone grafts could maintain good proliferation activity and osteogenic phenotype. The biological phenotype of these cells could be well preserved. The transplantation of cryopreserved bone-derived cells on scaffold could promote new bone formation in nude mice and enhance the osteointegration for dental implants in canine, which confirmed their osteogenic capacity, and showed that cells derived from cryopreserved bone were comparable to that of fresh bone in terms of the ability to promote osteogenesis in vivo. This work demonstrates that cryopreserved bone grafts may represent a novel, accessible source of cells for tissue regeneration therapy, and the results of our study may also stimulate the development of other cryopreservation techniques in basic and clinical studies.

Long-term outcome of cryopreserved bone-derived osteoblasts for bone regeneration in vivo.

Cryopreserved bone-derived osteoblasts (CBOs) have been considered as a promising cell source for bone regeneration. Previous studies have demonstrated that CBOs had good proliferation and osteogenicity. However, the long-term outcome of CBOs in vivo still remains unknown. In this experiment, we applied CBOs combined with calcium phosphate cement (CPC) to augment maxillary sinus in canine, computer tomography, polychrome labeling, biomechanical tests, fluorescent immunohistochemistry staining and histological analysis were used to analyze the property and mineralization process of the tissue-engineered bone preclinical application. Our results showed that CBOs combined with CPC could promote bone regeneration, dramatically maintain the height, volume and biomechanical property of augmented maxillary sinus. Furthermore, the tissue-engineered bone was more mature than scaffold alone or autogenous bone, and bone formation and remodeling were still apparent 20 months postoperatively. Additionally, 4 months after surgery might be the suitable time point for implants placement in the regenerated bone. These results also indicate that cryopreserved bone may be a potential source of osteoblasts for maxillary sinus augmentation.

Systematic evaluation of a tissue-engineered bone for maxillary sinus augmentation in large animal canine model.

The objective of this study is to systematically evaluate the effects of a tissue-engineered bone complex for maxillary sinus augmentation in a canine model. Twelve sinus floor augmentation surgeries in 6 animals were performed bilaterally and randomly repaired with the following 3 groups of grafts: group A consisted of tissue-engineered osteoblasts/beta-TCP complex (n=4); group B consisted of beta-TCP alone (n=4); group C consisted of autogenous bone obtained from iliac crest as a positive control (n=4). All dogs had uneventful healings following the surgery. Sequential polychrome fluorescent labeling, maxillofacial CT, microhardness tests, as well as histological and histomorphometric analyses indicated that the tissue-engineered osteoblasts/beta-TCP complex dramatically promoted bone formation and mineralization and maximally maintained the height and volume of elevated maxillary sinus. By comparison, both control groups of beta-TCP or autologous iliac bone showed considerable resorption and replacement by fibrous or fatty tissue. We thus conclude that beta-TCP alone could barely maintain the height and volume of the elevated sinus floor, and that the transplantation of autogenous osteoblasts on beta-TCP could promote earlier bone formation and mineralization, maximally maintain height, volume and increase the compressive strength of augmented maxillary sinus. This tissue engineered bone complex might be a better alternative to autologous bone for the clinical edentulous maxillary sinus augmentation.

Vertical alveolar ridge augmentation with beta-tricalcium phosphate and autologous osteoblasts in canine mandible.

A tissue-engineered bone has become a viable alternative to autologous bone for bone augmentation in atrophy alveolar ridge. The aim of the present study was to evaluate porous beta-tricalcium phosphate (beta-TCP) combined with autologous osteoblasts to augment edentulous alveolar ridge in a canine model. Autologous osteoblasts were expanded and combined with beta-TCP scaffold to fabricate a tissue-engineered bone. 12 bilateral alveolar ridge augmentation surgeries were carried out in 6 beagle dogs with the following 3 groups: beta-TCP/osteoblasts, beta-TCP alone and autogenous iliac bone control (n=4 per group). Sequential fluorescent labeling and radiographs were used to compare new bone formation and mineralization in each group. 24 weeks later, animals were sacrificed and non-decalcified and decalcified sections were evaluated histologically and histomorphometrically. Results indicated that the tissue-engineered bone dramatically enhanced new bone formation and mineralization, increase the new bone area, and maintain the height and thickness of the augmented alveolar ridge when compared with beta-TCP alone group. More importantly, the tissue-engineered bone achieved an elevated bone height and thickness comparable to that of autogenous iliac bone graft. This study demonstrated the potential of porous beta-TCP as a substrate for autogenous osteoblasts in bone tissue engineering for alveolar ridge augmentation.

Apatite-coated silk fibroin scaffolds to healing mandibular border defects in canines.

Tissue engineering has become a new approach for repairing bony defects. Highly porous osteoconductive scaffolds perform the important role for the success of bone regeneration. By biomimetic strategy, apatite-coated porous biomaterial based on silk fibroin scaffolds (SS) might provide an enhanced osteogenic environment for bone-related outcomes. To assess the effects of apatite-coated silk fibroin (mSS) biomaterials for bone healing as a tissue engineered bony scaffold, we explored a tissue engineered bony graft using mSS seeded with osteogenically induced autologous bone marrow stromal cells (bMSCs) to repair inferior mandibular border defects in a canine model. The results were compared with those treated with bMSCs/SS constructs, mSS alone, SS alone, autologous mandibular grafts and untreated blank defects. According to radiographic and histological examination, new bone formation was observed from 4 weeks post-operation, and the defect site was completely repaired after 12 months for the bMSCs/mSS group. In the bMSCs/SS group, new bone formation was observed with more residual silk scaffold remaining at the center of the defect compared with the bMSCs/mSS group. The engineered bone with bMSCs/mSS achieved satisfactory bone mineral densities (BMD) at 12 months post-operation close to those of normal mandible (p>0.05). The quantities of newly formed bone area for the bMSCs/mSS group was higher than the bMSCs/SS group (p<0.01), but no significant differences were found when compared with the autograft group (p>0.05). In contrast, bony defects remained in the center with undegraded silk fibroin scaffold and fibrous connective tissue, and new bone only formed at the periphery in the groups treated with mSS or SS alone. The results suggested that apatite-coated silk fibroin scaffolds combined with bMSCs could be successfully used to repair mandibular critical size border defects and the premineralization of these porous silk fibroin protein scaffolds provided an increased osteoconductive environment for bMSCs to regenerate sufficient new bone tissue.

Mandibular repair in rats with premineralized silk scaffolds and BMP-2-modified bMSCs.

Premineralized silk fibroin protein scaffolds (mSS) were prepared to combine the osteoconductive properties of biological apatite with aqueous-derived silk scaffold (SS) as a composite scaffold for bone regeneration. The aim of present study was to evaluate the effect of premineralized silk scaffolds combined with bone morphogenetic protein-2 (BMP-2) modified bone marrow stromal cells (bMSCs) to repair mandibular bony defects in a rat model. bMSCs were expanded and transduced with adenovirus AdBMP-2, AdLacZ gene in vitro. These genetically modified bMSCs were then combined with premineralized silk scaffolds to form tissue-engineered bone. Mandibular repairs with AdBMP-2 transduced bMSCs/mSS constructs were compared with those treated with AdLacZ-transduced bMSCs/mSS constructs, native (nontransduced) bMSCs/mSS constructs and mSS alone. Eight weeks after post-operation, the mandibles were explanted and evaluated by radiographic observation, micro-CT, histological analysis and immunohistochemistry. The presence of BMP-2 gene enhanced tissue-engineered bone in terms of the most new bone formed and the highest local bone mineral densities (BMD) found. These results demonstrated that premineralized silk scaffold could serve as a potential substrate for bMSCs to construct tissue-engineered bone for mandibular bony defects. BMP-2 gene therapy and tissue engineering techniques could be used in mandibular repair and bone regeneration.