Osseointegration of porous apatite-wollastonite and poly(lactic acid) composite structures created using 3D printing techniques.

A novel apatite-wollastonite/poly(lactic acid) (AW/PLA) composite structure, which matches cortical and cancellous bone properties has been produced and evaluated in vitro and in vivo. The composites structure has been produced using an innovative combination of 3D printed polymer and ceramic macrostructures, thermally bonded to create a hybrid composite structure. In vitro cell assays demonstrated that the AW structure alone, PLA structure alone, and AW/PLA composite were all biocompatible, with the AW structure supporting the proliferation and osteogenic differentiation of rat bone marrow stromal cells. Within a rat calvarial defect model the AW material showed excellent osseointegration with the formation of new bone, and vascularisation of the porous AW structure, both when the AW was implanted alone and when it was part of the AW/PLA composite structure. However, the AW/PLA structure showed the largest amount of the newly formed bone in vivo, an effect which is considered to be a result of the presence of the osteoinductive AW structure stimulating bone growth in the larger pores of the adjacent PLA structure. The layered AW/PLA structure showed no signs of delamination in any of the in vitro or in vivo studies, a result which is attributed to good initial bonding between polymer and ceramic, slow resorption rates of the two materials, and excellent osseointegration. It is concluded that macro-scale composites offer an alternative route to the fabrication of bioactive bone implants which can provide a match to both cortical and cancellous bone properties over millimetre length scales.

Preparation and characterisation of an innovative injectable calcium sulphate based bone cement for vertebroplasty application.

In this study, an innovative injectable and bioresorbable composite cement (Spine-Ghost) has been developed by combining a radiopaque glass-ceramic powder (SCNZgc) and spray-dried mesoporous bioactive particles (W-SC) into type III alpha calcium sulphate hemihydrate (α-CSH) (composition α-CSH/SCNZgc/W-SC, 70/20/10 wt%). The Spine-Ghost cement and pure α-CSH (as a reference) were characterised in terms of physical and mechanical properties and compared to a commercial reference (Cerament®- Bonesupport AB, Sweden). The Spine-Ghost cement had a setting time comparable with Cerament® showing a good injectability in the range of 8-20 minutes after the end of mixing. In addition, the Spine-Ghost cement showed a good radiopacity when compared with standard PMMA (BonOs Inject, aap Biomaterials GmbH Germany) and higher compressive strength when compared to healthy cancellous bone. The bioactivity of both Spine-Ghost and Cerament® was evaluated through in vitro soaking in simulated body fluid (SBF). Spine-Ghost samples were highly bioactive, inducing the precipitation of hydroxyapatite crystals in the first week of soaking in vitro. It was also found that the degradation kinetics of the Spine-Ghost cement were faster than those of pure α-CSH and comparable to those of Cerament® after approximately 1 month of soaking in SBF. Moreover, the Spine-Ghost cement was cytocompatible in indirect-contact culture in vitro. Overall results indicate that the Spine-Ghost cement might be a very good candidate for vertebroplasty application and could enhance new bone formation in vivo.

Development of a methacrylate-terminated PLGA copolymer for potential use in craniomaxillofacial fracture plates.

We synthesised methacrylate-terminated PLGA (HT-PLGA, 85:15 LA:GA, 169kDa), for potential use as an adhesively attached craniomaxillofacial fracture fixation plate. The in vitro degradation of molecular weight, pH and flexural modulus were measured over 6weeks storage in PBS at 37°C, with commercially available high (225kDa, H-PLGA) and low (116kDa, L-PLGA) molecular weight 85:15 PLGAs used as comparators. Molecular weights of the materials reduced over 6weeks, HT-PLGA by 48%, H-PLGA by 23% and L-PLGA by 81%. HT-PLGA and H-PLGA exhibited a near constant pH (7.35) and had average flexural moduli in excess of 6GPa when produced, similar to that of the mandible. After 1week storage both exhibited a significant reduction in average modulus, however, from weeks 1-6 no further significant changes were observed, the average modulus never dropped significantly below 5.5GPa. In contrast, the L-PLGA caused a pH drop to below 7.3 by week 6 and an average modulus drop to 0.6 from an initial 4.6GPa. Cell culture using rat bone marrow stromal cells, revealed all materials were cytocompatible and exhibited no osteogenic potential. We conclude that our functionalised PLGA retains mechanical properties which are suitable for use in craniofacial fixation plates.

Bone marrow stromal cells enhance the osteogenic properties of hydroxyapatite scaffolds by modulating the foreign body reaction.

We aimed to investigate the osteogenic properties of bone marrow stromal cell (BMSC)-loaded biomimetic constructs composed of hydroxyapatite (HA), with or without in vitro cell-derived extracellular matrix (HA-ECM), and to assess the cellular components of the elicited foreign body reaction. HA-ECM constructs were produced by adult rat dermal fibroblasts cultured on top of synthetic HA microparticles. Rat calvarial critical-sized defects (8 mm) were created and treated with the generated HA-ECM constructs or HA microparticles, alone or combined with green fluorescent protein (GFP)-expressing BMSCs. The new bone formation and the local cellular inflammatory response (macrophages, neutrophils, lymphocytes, eosinophils and PCNA-index) were assessed by histomorphometry and immunohistochemistry at 2 and 12 weeks postoperatively. In addition, the BMSCs' survival and engraftment were checked. The largest volume of the newly formed bone was found in defects treated with HA-ECM constructs combined with BMSCs (p < 0.05). Moreover, the implanted BMSCs modulated the local inflammatory response, demonstrated by either a significant increase (HA vs HA + BMSCs) or decrease (HA-ECM vs HA-ECM + BMSCs) of the inflammatory cell number. No donor BMSCs were detected at the site of implantation or in the host bone marrow at 2 or 12 weeks postoperatively. In conclusion, the treatment of critical-sized calvarial defects with the BMSC-loaded biomimetic constructs has significantly enhanced bone repair by modulating the foreign body reaction. Our findings highlight the implications of BMSCs in the regulation of the foreign body reaction triggered by tissue-engineered constructs, proving a higher efficiency for the BMSC combination therapy.

Human fibroblast-derived extracellular matrix constructs for bone tissue engineering applications.

We exploited the biomimetic approach to generate constructs composed of synthetic biphasic calcium phosphate ceramic and extracellular matrix (SBC-ECM) derived from adult human dermal fibroblasts in complete xeno-free culture conditions. The construct morphology and composition were assessed by scanning electron microscopy, histology, immunohistochemistry, Western blot, glycosaminoglycan, and hydroxyproline assays. Residual DNA quantification, endotoxin testing, and local inflammatory response after implantation in a rat critical-sized calvarial defect were used to access the construct biocompatibility. Moreover, in vitro interaction of human mesenchymal stem cells (hMSCs) with the constructs was studied. The bone marrow- and adipose tissue-derived mesenchymal stem cells were characterized by flow cytometry and tested for osteogenic differentiation capacity prior seeding onto SBC-ECM, followed by alkaline phosphatase, 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, and real-time quantitative polymerase chain reaction to assess the osteogenic differentiation of hMSCs after seeding onto the constructs at different time intervals. The SBC-ECM constructs enhanced osteogenic differentiation of hMSCs in vitro and exhibited excellent handling properties and high biocompatibility in vivo. Our results highlight the ability to generate in vitro fibroblast-derived ECM constructs in complete xeno-free conditions as a step toward clinical translation, and the potential use of SBC-ECM in craniofacial bone tissue engineering applications.

Transplanted Human Bone Marrow Mesenchymal Stem Cells Seeded onto Peptide Hydrogel Decrease Alveolar Bone Loss.

Alveolar bone loss can be caused by periodontitis or periodontal trauma. We have evaluated the effects of transplanted undifferentiated human mesenchymal stem cells (hMSCs) on alveolar bone reaction and periodontal ligament healing in an experimental periodontal wound model. The hMSCs seeded onto a self-assembling peptide hydrogel in combination with collagen sponge were implanted into the right mandible of 12 rats and followed for 1 (n=6) or 4 weeks (n=6) postoperatively. The other 12 sham-treated rats were used as controls. Histological and histomorphometrical methods were used to assess the periodontal tissue reaction. The alveolar bone volume density was significantly higher at 1 week after surgery, and the osteoclast number was significantly lower at both 1 week and 4 weeks postoperatively in the mandibles treated with hMSCs. The implanted cells were detected only at 1 week after surgery. In conclusion, transplanted hMSCs can contribute to alveolar bone preservation after a periodontal surgical trauma at least by decreasing local osteoclast number.

Cell-derived matrix enhances osteogenic properties of hydroxyapatite.

The study aimed to evaluate osteogenic properties of hydroxyapatite (HA) scaffold combined with extracellular matrix (ECM) derived in vitro from rat primary calvarial osteoblasts or dermal fibroblasts. The cellular viability, and the ECM deposited onto synthetic HA microparticles were assessed by MTT, Glycosaminoglycan, and Hydroxyproline assays as well as immunohistochemistry and scanning electron microscopy after 21 days of culture. The decellularized HA-ECM constructs were implanted in critical-sized calvarial defects of Sprague-Dawley rats, followed by bone repair and local inflammatory response assessments by histomorphometry and immunohistochemistry at 12 weeks postoperatively. We demonstrated that HA supported cellular adhesion, growth, and ECM production in vitro, and the HA-ECM constructs significantly enhanced calvarial bone repair (p<0.05, Mann-Whitney U-test), compared with HA alone, despite the significantly increased number of CD68+ macrophages, and foreign body giant cells (p<0.05, Mann-Whitney U-test). Selective accumulation of bone sialoprotein, osteopontin, and periostin was observed at the tissue-HA interfaces. In conclusion, in vitro-derived ECM mimics the native bone matrix, enhances the osteogenic properties of the HA microparticles, and might modulate the local inflammatory response in a bone repair-favorable way. Our findings highlight the ability to produce functional HA-ECM constructs for bone tissue engineering applications.

Early timing of low-dose dexamethasone decreases inflammation in a murine model of eosinophilic airway disease.

CONCLUSION: A very low dose of dexamethasone (DEX) was as equally as sufficient as a pharmacological dose to decrease eosinophil inflammation in airways and bone marrow. The timing of DEX treatment in relation to allergen challenge was strongly decisive for the outcome of the inflammatory response. OBJECTIVES: We aimed to study compartmental allergic airway inflammatory responses to classic pharmacological and also extremely low physiological DEX dosage, given at different time points close to allergen challenge in a murine model. MATERIALS AND METHODS: Ovalbumin-sensitized BALB/c-mice were exposed to intra-nasal ovalbumin. DEX was given i.p. as 1 microg/kg low-dose or 500 microg/kg pharmacological single-dose 2 h before, immediately before or 7 h after each of three challenges. Inflammatory cells were evaluated in bronchoalveolar lavage (BAL), lungs, nasal mucosa, and bone marrow. RESULTS: Groups treated with low-dose DEX decreased eosinophilia in BAL to the same extent as the pharmacological dose, but only when administered before challenge. The most prominent decrease of eosinophils in BAL was seen in mice treated with the low dose 2 h before challenge. A similar response pattern as in BAL eosinophilia was detected in lung histopathology. DEX treatments had no obvious effects on inflammation in nasal mucosa.

Collagen-hydroxyapatite composite enhances regeneration of calvaria bone defects in young rats but postpones the regeneration of calvaria bone in aged rats.

The potential differences in bone repair of calvaria defects treated with a collagen sponge (HELISTAT) or a collagen-hydroxyapatite composite (HEALOS) in young and aged rats were evaluated at 8 weeks after surgery. A histomorphometric analysis of new bone formation and an evaluation of angiogenesis, mast cell, and eosinophil local infiltration were performed. Evaluation showed that HELISTAT induced a similar amount of new bone in both young and aged rats. However this occurred to a lesser degree than in young rats treated with HEALOS. The largest number of blood vessels was present in the defects of aged rats treated with HEALOS, and the number of mast cells was highest in the defects treated with HELISTAT in both young and aged rats. Eosinophils were present to the greatest extent in defects treated with HEALOS in comparison to defects treated with HELISTAT in both young and aged rats. Collagen-hydroxyapatite composite (HEALOS) enhances calvarial bone repair more than collagen sponge alone (HELISTAT) in young rats but not in aged rats at 8 weeks after surgery. HEALOS appears to induce a more intense inflammatory response than HELISTAT especially in aged rats.

Local treatment of cricoid cartilage defects with rhBMP-2 induces growth plate-like morphology of chondrogenesis.

OBJECTIVE: The ultrastructural characteristics of new bone and cartilage, induced at the site of cricoid cartilage defects treated with rhBMP-2 in rabbits, were investigated. STUDY DESIGN AND SETTING: A cricoid defect model was used. Fifteen rabbits were randomly and equally divided into 3 groups. Four rabbits from each group were treated with rhBMP-2, while one rabbit from each group was used as control. The rabbits were killed 1, 2, or 4 weeks after surgery. The healing pattern of the laryngeal wound was evaluated by light and transmission electron microscopy. RESULTS: Mineralized collagen type I matrix, osteoblasts, and osteoclast-like cells were present as early as 1 week after surgery. Well-structured bone trabeculas and growth plate-like structures were present 4 weeks after surgery. CONCLUSION: Intramembranous and endochondral osteogenesis take place at the site of cricoid cartilage defects treated with rhBMP-2. Progenitor cells of cricoid perichondrium form a growth plate-like structure similar to the epiphyseal growth plate. SIGNIFICANCE: This study reveals the pattern of BMP-2-induced repair of airway cartilage defects.

Cell origin in experimental repair of cricoid cartilage defects treated with recombinant human bone morphogenetic protein-2.

We determined the origin of new cartilage and new bone induced by recombinant human bone morphogenetic protein-2 (rhBMP-2) at the site of cricoid cartilage defects in rabbits randomly divided into eight groups. The cricoid cartilage was split vertically along the anterior midline and a strip was excised from the anterior part of the cricoid cartilage in all rabbits. The perichondrium from the anterior part of the cricoid cartilage was trimmed off in four groups; two groups treated with rhBMP-2 and two control groups. In four other groups, the anterior perichondrium was detached and used as a flap with two groups treated with rhBMP-2 and two groups serving as controls. The rabbits were killed 1 week or 4 weeks after surgery. The larynges were removed, fixed and sectioned, and the sections were stained for light microscopy using various cytochemical and immunological techniques. New cartilage was only present close to the host perichondrium adherent to cricoid cartilage in rabbits treated with rhBMP-2. New bone was present 4 weeks after surgery, although calcified matrix and alkaline phosphatase activity could be detected at the site of cricoid defects as early as 1 week after surgery. The cell proliferation marker Ki-67 was strongly expressed in granulation tissue and bone marrow, and it was moderately expressed in muscles adjacent to the cricoid cartilage in rhBMP-2-treated specimens. BMP receptors were strongly expressed in cartilage and moderately expressed in adjacent muscles. We conclude that new cartilage originates from the mesenchymal progenitor cells of host perichondrium adherent to cricoid cartilage in rabbits treated with rhBMP-2. New bone may originate from local muscle.

Effect of recombinant human BMP-2 on the repair of cricoid cartilage defects in young and adult rabbits: a comparative study.

OBJECTIVE: The study evaluated the possible differences in the repair of cricoid cartilage defects treated with recombinant human BMP-2 in young and adult rabbits. METHODS: A cricoid defect rabbit model was used. Thirty rabbits were randomly divided into eight groups. Two groups of young rabbits and two groups of adult rabbits were treated with rhBMP-2 delivered on an absorbable collagen sponge, while the other two groups of young rabbits and two groups of adult rabbits were used as controls. The rabbits were killed at 1 week or 4 weeks after surgery. A histomorphometric analysis and an evaluation of the expression of collagen types I, II, and X, and proliferating cell nuclear antigen as well as a study of distribution of calcified matrix, were performed. RESULTS: rhBMP-2 induced a marked chondrogenesis in both experimental age groups. However, in young rabbits the newly formed cartilage appeared more elongate, and the length of perichondrium involved was greater. The host cricoid cartilage of adult rabbits was calcified in large areas and displayed a strong matrix expression of collagen type X as well as collagen type I in the perichondrium, compared to the cricoid of young rabbits. In spite of these differences no immunohistochemical differences were found in the newly formed cartilage of both age groups treated with rhBMP-2. The cricoid cartilage defect was filled with new bone at 4 weeks in both age groups treated with rhBMP-2. New bone tissue had a well-defined trabecular structure. CONCLUSIONS: rhBMP-2 triggers appositional cartilage growth from the cricoid perichondrium of young rabbits more easily than from that of adult rabbits. The new bone induced by rhBMP-2 showed a similar immunohistochemical and morphological pattern in both age groups of rabbits.

Recombinant human bone morphogenetic protein-2 enhances experimental laryngotracheal reconstruction in rabbits.

OBJECTIVE: Bone morphogenetic protein-2 offers potential benefits for cartilage regeneration. We investigated the effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) on the regeneration of laryngeal cartilage and respiratory epithelium in a rabbit model. MATERIAL AND METHODS: We used a cricoid defect rabbit model. Twenty-four rabbits were randomly divided into four equal groups. Two groups were treated with 5 microg of rhBMP-2 delivered on an absorbable collagen sponge and the other two groups were used as controls. One group of treated rabbits and one group of control rabbits were euthanized 1 week after surgery, while the others were euthanized 4 weeks after surgery. The healing pattern of the laryngeal wound was evaluated by means of histomorphometry. RESULTS: Regeneration of both the epithelial layer and cartilage was significantly better in rabbits treated with rhBMP-2. Four weeks after surgery, the cricoid cartilage defect was completely repaired by new cartilage and new bone in rabbits treated with rhBMP-2. Furthermore, the lining respiratory epithelium healed more rapidly in treated rabbits. CONCLUSION: rhBMP-2, delivered via an absorbable collagen sponge, induces complete regeneration and repair of rabbit cricoid cartilage defects. It also induces faster relining and regeneration of airway epithelium than in control rabbits.

Structural characteristics of repair tissue of cricoid cartilage defects treated with recombinant human bone morphogenetic protein-2.

We examined the structural characteristics of repair tissue induced by recombinant human bone morphogenetic protein-2 in a rabbit model of laryngotracheal reconstruction. Twenty-four New Zealand White rabbits were randomly divided into four groups of six rabbits. Two groups were treated with recombinant human bone morphogenetic protein-2 delivered on an absorbable collagen sponge, while two groups were used as controls. Rabbits were euthanized at 1 and 4 weeks after surgery. The larynx was removed, fixed, and sectioned. The sections were stained with hematoxylin-eosin, safranine O/fast green, and immunostained with an antibody for tissue inhibitor of metalloproteinases-1. In rabbits treated with bone morphogenetic protein-2, the defects were filled with new cartilage and bone at 4 weeks after surgery. There were no discontinuities or gaps at the margins of the cartilage defects. Proteoglycans were synthesized in new cartilage in rabbits treated with bone morphogenetic protein-2, and were present 4 weeks after surgery. The general aspects of the vascular pattern and the pattern of tissue inhibitor of metalloproteinases-1 expression were similar in control and treated rabbits, both 1 week and 4 weeks after surgery. The repair tissue induced by recombinant human bone morphogenetic protein-2 consisted of new cartilage and bone perfectly integrated with host tissue at the site of the cricoid cartilage defects. This new cartilage was able to mature and produce proteoglycans.

Mifepristone (RU-486) impairs post-surgical wound healing of the larynx.

BACKGROUND: The purpose of this study was to examine how RU486 (mifepristone), a glucocorticoid antagonist, affects the regeneration process in a rabbit laryngeal wound-healing model. MATERIAL/METHODS: Eight rabbits underwent a cricoid-split operation with collagen sponge insertion. The animals were classified randomly into two groups (4 animals in each group): the RU486-treated group, with local administration of 3 mg RU486, and the control group, which was sham treated. The specimens were harvested seven days after surgery. Histomorphometric analysis was performed to evaluate the healing pattern of the larynx wound. RESULTS: Respiratory epithelium regeneration was delayed in the RU486-treated group. The proliferation of granulation tissue was also increased. Polyps were detected in 3 specimens from this group. No obvious differences with regard to inflammatory cell infiltration were found. CONCLUSIONS: RU486 impairs post-surgical wound healing of the larynx by increasing the presence of granulation tissue and polyps, and appears to delay re-epithelialization.