In vitro assessment of connection strength and stability of internal implant-abutment connections.

BACKGROUND: Various connections have been machined to improve the fit between the dental abutment and implant. In vivo, the instability created by imprecisely fitting components can cause soft tissue irritation and bacterial colonization of the implant system. The aim of this study was to quantify abutment stability under in vitro force applications. METHODS: Abutment stability and fit were quantitatively measured after application of rotational, vertical, and horizontal forces. FINDINGS: The abutment connection held by friction (Friction-Fit) was the only group to have 0° angular rotation. A significantly greater vertical force was required to pull the abutment from the implant for the Friction-Fit connection as compared to all other experimental groups. The abutment connection held by a mechanically locking friction-fit with four grooves (CrossFit) and Friction-Fit demonstrated significantly lower lateral movement as compared to all other connections. The remaining connections evaluated included two hexagon connections that rely on screw placement for abutment fit (Conical + Hex #1 and Conical + Hex #2), one connection with protruding slots to align with recessed channels inside the implant (Conical + 6 Indexing Slots), and an internal connection that allows for abutment indexing every 120° (Internal Tri-Channel). INTERPRETATION: Internal connection geometry influenced the degree of abutment movement. Friction-Fit and CrossFit connections exhibited the lowest rotational and horizontal motions. Significant differences were found between Friction-Fit and CrossFit following the application of a vertical force, with the Friction-Fit requiring a significantly greater pull force to separate the abutment from the implant.

Chitosan-poly(lactide-co-glycolide) microsphere-based scaffolds for bone tissue engineering: in vitro degradation and in vivo bone regeneration studies.

Natural polymer chitosan and synthetic polymer poly(lactide-co-glycolide) (PLAGA) have been investigated for a variety of tissue engineering applications. We have previously reported the fabrication and in vitro evaluation of a novel chitosan/PLAGA sintered microsphere scaffold for load-bearing bone tissue engineering applications. In this study, the in vitro degradation characteristics of the chitosan/PLAGA scaffold and the in vivo bone formation capacity of the chitosan/PLAGA-based scaffolds in a rabbit ulnar critical-sized-defect model were investigated. The chitosan/PLAGA scaffold showed slower degradation than the PLAGA scaffold in vitro. Although chitosan/PLAGA scaffold showed a gradual decrease in compressive properties during the 12-week degradation period, the compressive strength and compressive modulus remained in the range of human trabecular bone. Chitosan/PLAGA-based scaffolds were able to guide bone formation in a rabbit ulnar critical-sized-defect model. Microcomputed tomography analysis demonstrated that successful bridging of the critical-sized defect on the sides both adjacent to and away from the radius occurred using chitosan/PLAGA-based scaffolds. Immobilization of heparin and recombinant human bone morphogenetic protein-2 on the chitosan/PLAGA scaffold surface promoted early bone formation as evidenced by complete bridging of the defect along the radius and significantly enhanced mechanical properties when compared to the chitosan/PLAGA scaffold. Furthermore, histological analysis suggested that chitosan/PLAGA-based scaffolds supported normal bone formation via intramembranous formation.

Bone tissue engineering by gene delivery.

Recombinant human bone morphogenetic protein-2 and -7 were recently granted United States Food and Drug Administration approval for select clinical applications in bone repair. While significant progress has been made in the delivery of recombinant osteogenic factor to promote bone healing, the short half-life and instability of the protein requires the delivery of milligram quantities of factor or multiple dosages. The potential of gene therapy for bone regeneration is the delivery of physiological levels of therapeutic protein using natural cellular mechanisms. Experimental investigations have demonstrated this approach uses lower dosages of factor to yield bone healing equivalent to that achieved via the administration of recombinant factor or use of bone grafts. The current states of gene delivery for bone tissue engineering applications and challenges to be met are presented in this review. Over the past couple of years, studies have continued to examine the delivery of the osteogenic factor bone morphogenetic protein using gene therapies. The importance of angiogenesis to bone formation has prompted the development of vascular endothelial growth factor gene expression systems for bone regeneration. Viral vectors, in combination with allograft bone, have been investigated to improve existing surgical care. Newly constructed vectors with reduced immunogenicity and regulated gene expression systems provide a greater degree of control over the timing and level of gene expression. Several advances have allowed bone tissue engineering by gene delivery to advance beyond serving as a potential treatment for isolated bone defects and fractures to a gene therapy approach for the treatment of genetic based bone diseases, such as osteogenesis imperfecta.

Human osteoblast cells: isolation, characterization, and growth on polymers for musculoskeletal tissue engineering.

We performed a detailed examination of the isolation, characterization, and growth of human osteoblast cells derived from trabecular bone. We further examined the morphology, phenotypic gene expression, mineralization,and growth of these human osteoblasts on polyester polymers used for musculoskeletal tissue engineering. Polylactic-co-glycolic acid [PLAGA (85:15, 50:50, 75:25)], and poly-lactic acid (L-PLA, D,L-PLA) were examined. The osteoblastic expression of key phenotypic markers osteocalcin, alkaline phosphatase, collagen, and bone sialoprotein at 4 and 8 weeks was examined. Reverse transcription-polymerase chain reaction studies revealed that trabecular-derived osteoblasts were positive for all markers evaluated with higher levels expressed over long-term culture. These cells also revealed mineralization and maturation as evidenced by energy dispersive X-ray analysis and scanning electron microscopy. Growth studies on PLAGA at 50:50,75:25, and 85:15 ratios and PLA in the L and DL isoforms revealed that human osteoblasts actively grew, with significantly higher cell numbers attached to scaffolds composed of PLAGA 50:50 in the short term and PLAGA 85:15 in the long term compared with PLA (p < 0.05). We believe human cell adhesion among these polymeric materials may be dependent on differences in cellular integrin expression and extracellular matrix protein elaboration.

Evaluation of the anterior cruciate ligament, medial collateral ligament, achilles tendon and patellar tendon as cell sources for tissue-engineered ligament.

This study investigated four different connective tissue cell types to determine which cell type should be the source for seeding a tissue-engineered anterior cruciate ligament (ACL) replacement. Cells derived from the ACL, medial collateral ligament (MCL), achilles tendon (AT), and patellar tendon (PT) of New Zealand White rabbits were isolated and cultured. Each cell type was cultured in vitro after seeding on three-dimensional (3-D) braided polymer scaffolds and on tissue culture polystyrene that served as a control. Samples were evaluated and compared for their morphology, proliferation, and gene expression of fibronectin, type I and type III collagen. Scanning electron microscopy (SEM) photomicrographs verified cell attachment of all four types of connective tissue fibroblasts to the scaffolds. Preliminary results comparing proliferation indicate that cells obtained from the PT and AT have the fastest proliferation. Whereas gene expression of the phenotypic markers measured using real-time reverse transcription polymerase chain reaction (RT-PCR) indicates ACL cells have the highest gene expression for the matrix markers. This leads to the question of which cell type should be the cell source for tissue-engineering of ligament, the highly proliferating cells or the differentiated matrix producing cells. This study would suggest that ACL differentiated matrix producing cells are the most suitable cells for further study and development of a tissue-engineered ligament.

Molecular regulation of osteoblasts for tissue engineered bone repair.

The use of biodegradable polymers in medicine and biomedical research is increasing. A key growth area has been the use of these materials in tissue engineering, especially for guided regeneration of bone and cartilage. Our interest has been in determining the mechanisms by which cellular attachment and growth occurs on these materials. In the current study, we examined human osteoblast cell adhesion, growth, and morphologic changes on polymeric scaffolds composed of polylactic-co-glycolic acid and polylactic acid materials. We examined these characteristics in association with measurements of levels of key adhesion integrin receptors in the presence and absence of antibodies against alpha2, alpha3, alpha4, alpha5, alpha6, and beta1 subunits, and the adhesion ligand peptides RGD (Arg-Gly-Asp) and RGE (Arg-Gly-Ser). At 2 hours, results showed initial cell adhesion was considerably decreased on polylactic-co-glycolic acid and polylactic acid in the presence of the alpha2 and beta1, antibodies with a 70% adhesion rate difference observed among the groups evaluated. Higher levels of inhibition were observed on polylactic-co-glycolic acid relative to polylactic acid, which may be correlated to a higher number of cells being able to interact with the surface initially. The presence of known competitive peptide (RGD) at 2 hours, revealed its ability to block cellular adhesion to these matrices relative to the control and noncompetitive peptide RGE on polylactic-co-glycolic acid matrices. Overall adhesion rate was affected by the presence of the integrin antibodies to the alpha2, alpha3, alpha4, alpha5, alpha6, and beta1 subunits with highest differences among polylactic-co-glycolic acid relative to its control, therefore suggesting that initial osteoblastic cell adhesion to commonly used biomaterials is regulated through integrin binding.

Protein- and gene-based tissue engineering in bone repair.

A tissue engineering approach to bone regeneration includes the use of a scaffold, cells and bioactive factors alone or in various combinations. Several investigators have demonstrated enhanced bone formation when the tissue-engineered construct possesses traits inherent to autogenic bone grafts, namely osteoconductivity, osteoinductivity and osteogenicity. Use of the biodegradable polymer poly(lactide-co-glycolide) in combination with bone morphogenetic protein or primary cells genetically modified to release osteogenic protein have demonstrated the ability to induce osteogenic differentiation of, and subsequent mineralization by, muscle-derived cells and mesenchymal stem cells in both in vitro and in vivo applications.

Development of a calcium phosphate co-precipitate/poly(lactide-co-glycolide) DNA delivery system: release kinetics and cellular transfection studies.

One of the most common non-viral methods for the introduction of foreign deoxyribonucleic acid (DNA) into cultured cells is calcium phosphate co-precipitate transfection. This technique involves the encapsulation of DNA within a calcium phosphate co-precipitate, particulate addition to in vitro cell culture, endocytosis of the co-precipitate, and exogenous DNA expression by the transfected cell. In this study, we fabricated a novel non-viral gene transfer system by adsorbing DNA, encapsulated in calcium phosphate (DNA/Ca-P) co-precipitates, to biodegradable two- and three-dimensional poly(lactide-co-glycolide) matrices (2D-DNA/Ca-P/PLAGA, 3D-DNA/Ca-P/PLAGA). Co-precipitate release studies demonstrated an initial burst release over the first 48 h. By day 7, approximately 96% of the initially adsorbed DNA/Ca-P co-precipitate had been released. This was followed by low levels of co-precipitate release for 42 days. Polymerase chain reaction was used to demonstrate the ability of the released DNA containing co-precipitates to transfect SaOS-2 cells cultured in vitro on the 3D-DNA/Ca-P/PLAGA matrix and maintenance of the structural integrity of the exogenous DNA. In summary, a promising system for the incorporation and controlled delivery of exogenous genes encapsulated within a calcium phosphate co-precipitate from biodegradable polymeric matrices has been developed and may have applicability to the delivery of therapeutic genes and the transfection of other cell types.

Cryopreservation of tissue engineered constructs for bone.

The large-scale clinical use of tissue engineered constructs will require provisions for its mass availability and accessibility. Therefore, it is imperative to understand the effects of low temperature (-196 degrees C) on the tissue engineered biological system. Initial studies used samples of the osteoblast-like cell line (SaOS-2) adhered to a two-dimensional poly(lactide-co-glycolide) thin film (2D-PLAGA) or a three-dimensional poly(lactide-co-glycolide) sintered microsphere matrix (3D-PLAGA) designed for bone tissue engineering. Experimental samples were tested for their ability to maintain cell viability, following low temperature banking for one week, in solutions of the penetrating cryoprotective agents, dimethylsulfoxide (DMSO), ethylene glycol, and glycerol. Results indicated the DMSO solution yielded the greatest percent cell survival for SaOS-2 cells adhered to both the 2D- and 3D-PLAGA scaffolds; therefore, DMSO was used to cryopreserve mineralizing primary rabbit osteoblasts cells adhered to 2D-PLAGA matrices for 35 days. Results indicated retention of the extracellular matrix architecture as no statistically significant difference in the pre- and post-thaw mineralized structures was measured. Percent cell viability of the mineralized constructs following low temperature storage was approximately 50%. These are the first studies to address the issue of preservation techniques for tissue engineered constructs. The ability to successfully cryopreserve mineralized tissue engineered matrices for bone may offer an unlimited and readily available source of bone-like materials for orthopaedic applications.

In vitro bone formation using muscle-derived cells: a new paradigm for bone tissue engineering using polymer-bone morphogenetic protein matrices.

Over 800,000 bone grafting procedures are performed in the United States annually, creating a demand for viable alternatives to autogenous bone, the grafting standard in osseous repair. The objective of this study was to examine the efficacy of a BMP-polymer matrix in inducing the expression of the osteoblastic phenotype and in vitro bone formation by muscle-derived cells. Specifically, we evaluated the ability of bone morphogenetic protein-7 (BMP-7), delivered from a poly(lactide-co-glycolide) (PLAGA) matrix, to induce the differentiation of cells derived from rabbit skeletal muscle into osteoblast-like cells and subsequently form mineralized tissue. Results confirmed that muscle-derived cells attached and proliferated on the PLAGA substrates. BMP-7 released from PLAGA induced the muscle-derived cells to increase bone marker expression and form mineralized cultures. These results demonstrate the efficacy of a BMP-polymer matrix in inducing the expression of the osteoblastic phenotype by muscle-derived cells and present a new paradigm for bone tissue engineering.