David A. Puleo

Understanding and controlling the bone–implant interface

A goal of current implantology research is to design devices that induce controlled, guided, and rapid healing. In addition to acceleration of normal wound healing phenomena, endosseous implants should result in formation of a characteristic interfacial layer and bone matrix with adequate biomechanical properties. To achieve these goals, however, a better understanding of events at the interface and of the effects biomaterials have on bone and bone cells is needed. Such knowledge is essential for developing strategies to optimally control osseointegration. This paper reviews current knowledge of the bone-biomaterial interface and methods being investigated for controlling it. Morphological studies have revealed the heterogeneity of the bone-implant interface. One feature often reported, regardless of implant material, is an afibrillar interfacial zone, comparable to cement lines and laminae limitantes at natural bone interfaces. These electron-dense interfacial layers are rich in noncollagenous proteins, such as osteopontin and bone sialoprotein. Several approaches, involving alteration of surface physicochemical, morphological, and/or biochemical properties, are being investigated in an effort to obtain a desirable bone-implant interface. Of particular interest are biochemical methods of surface modification, which immobilize molecules on biomaterials for the purpose of inducing specific cell and tissue responses or, in other words, to control the tissue-implant interface with biomolecules delivered directly to the interface. Although still in its infancy, early studies indicate the value of this methodology for controlling cell and matrix events at the bone-implant interface.

Calcium sulfate: Properties and clinical applications.

Calcium sulfate (CS) has enjoyed a longer history of clinical use than most currently available biomaterials. It is well-tolerated when used to fill bone defects and undergoes rapid and complete resorption without eliciting a significant inflammatory response. The raw material from which it is made is relatively inexpensive and abundant. In addition, CS can be used as a vehicle to deliver antibiotics, pharmacologic agents, and growth factors. It has found wide use in orthopedics and dentistry, and has been used in a variety of clinical applications, including the periodontal defect repair, the treatment of osteomyelitis, sinus augmentation, and as an adjunct to dental implant placement. Despite these advantages, the material has not enjoyed the popularity of many other regenerative materials, although there has been a recent resurgence of interest in the material. This review examines the properties and clinical applications of CS, with an emphasis on dental applications of the material. Limitations of the material are discussed as well as suggestions for future research.

In vitro effects of combined and sequential delivery of two bone growth factors

Bone formation and repair occur by a complex cascade involving numerous growth factors and cytokines. In this study, two-layered heterogeneously loaded and crosslinked gelatin coatings were used to obtain combined and sequential delivery of two bone growth factors, BMP-2 and IGF-I, in cell cultures. Peak release from the top and bottom layers was localized around 1 and 6 days, respectively. For comparison, cells were also treated with soluble growth factors directly added to the culture medium. Pluripotent C3H10T1/2 (C3H) cells responded to soluble growth factor treatments with the greatest specific alkaline phosphatase (AP) activity resulting from addition of BMP-2 followed by IGF-I or by BMP-2+IGF-I. Altered loading and subsequent release of BMP-2 and IGF-I from gelatin coatings also affected AP activity in C3H cultures, and the coatings influenced AP activity and incorporation of calcium in the extracellular matrix of bone marrow stromal cell cultures. Early delivery of BMP-2 followed by increased release of BMP-2 and IGF-I after 5 days resulted in the largest, as well as earliest, elevation of AP activity and mineralized matrix formation compared to controls and other treatments. Simultaneous release of both growth factors from both layers did not significantly change AP activity or matrix calcium content compared to control coatings. These results demonstrate that temporally varying delivery of multiple growth factors can significantly affect cell behavior.

Ti-6Al-4V ion solution inhibition of osteogenic cell phenotype as a function of differentiation timecourse in vitro.

Metal ions released from the implant surface are suspected of playing some contributing role in loosening of hip and knee prostheses. previous work in this laboratory demonstrated that sublethal doses of the ionic constituents of Ti-6Al-4V alloy suppressed expression of the osteoblastic phenotype and deposition of a mineralized matrix. The purpose of this work was to further explore this suppression as a function of the normal time-course of phenotype expression. Bone marrow stromal cells were harvested from juvenile rats and exposed to time-staggered doses of a solution of ions representing Ti-6Al-4V alloy. Cells were cultured for four weeks and assayed for total protein, alkaline phosphatase, intra-and extracellular osteocalcin, and calcium. Ti-6Al-4V solutions were found to produce little difference from control solutions for total protein or alkaline phosphatase levels, but strongly inhibited osteocalcin synthesis. Calcium levels were reduced when ions were added before a critical point of osteoblastic differentiation (between 2 and 3 weeks after seeding). These results indicate that ions associated with Ti-6Al-4V alloy inhibited the normal differentiation of bone marrow stromal cells to mature osteoblasts in vitro, suggesting that ions released from implants in vivo may contribute to implant failure by impairing normal bone deposition.

Animal Models for Periodontal Disease

Animal models and cell cultures have contributed new knowledge in biological sciences, including periodontology. Although cultured cells can be used to study physiological processes that occur during the pathogenesis of periodontitis, the complex host response fundamentally responsible for this disease cannot be reproduced in vitro. Among the animal kingdom, rodents, rabbits, pigs, dogs, and nonhuman primates have been used to model human periodontitis, each with advantages and disadvantages. Periodontitis commonly has been induced by placing a bacterial plaque retentive ligature in the gingival sulcus around the molar teeth. In addition, alveolar bone loss has been induced by inoculation or injection of human oral bacteria (e.g., Porphyromonas gingivalis) in different animal models. While animal models have provided a wide range of important data, it is sometimes difficult to determine whether the findings are applicable to humans. In addition, variability in host responses to bacterial infection among individuals contributes significantly to the expression of periodontal diseases. A practical and highly reproducible model that truly mimics the natural pathogenesis of human periodontal disease has yet to be developed.

Osteoblasts on hydroxyapatite, alumina and bone surfaces in vitro; morphology during the first 2 h of attachment

The morphological responses of individual osteoblasts as they attached and spread on hydroxyapatite, bovine bone, alumina with rough and polished surfaces, and tissue culture polystyrene in vitro were examined with scanning electron microscopy. Depending on the surface tested two different morphological sequences were observed during 2 h of adhesion. On alumina, both rough and smooth, bone, and tissue culture polystyrene the cells were round after 0.5 h, and spread radially during the next 1.5 h until they were almost flat, with a nuclear bulge. On hydroxyapatite, however, the cells were flat and circular at 0.5 h, and the edge of the cytoplasm was hardly discernable. This morphology did not change much during the subsequent 1.5 h. The observed cellular morphological response may be related to the bioreactivity of hydroxyapatite.

RGDS tetrapeptide binds to osteoblasts and inhibits fibronectin-mediated adhesion

The mechanisms of osteoblast attachment to surfaces were probed using the adhesive tetrapeptide RGDS (Arg-Gly-Asp-Ser) and the related but non-adhesive RGES (Arg-Gly-Glu-Ser). Specifically, RGDS and RGES were investigated for their ability both to bind to a suspension of well-characterized neonatal rat calvarial osteoblasts and to inhibit cell attachment to fibronectin-coated microtiter plates. RGDS bound to the cells with an average Kd approximately 9.4 x 10(-4) M, and RGES bound with an average Kd approximately 3.0 x 10(-4) M; at saturation, the osteoblasts bound almost twice as much RGDS as RGES. RGDS partially inhibited cell adhesion (55% to 60%) in a competitive, dose-dependent manner. In contrast, RGES had minimal effect on cell attachment. Since complete inhibition of attachment was not observed, it is likely that a synergistic adhesion site in the fibronectin molecule and/or cell surface molecules such as proteoglycans are active in mediating osteoblast/substrate adhesion.

Dependence of mesenchymal cell responses on duration of exposure to bone morphogenetic protein-2 in vitro.

Bone morphogenetic proteins (BMPs) induce osteoblastic responses in cultures of pluripotent mesenchymal cells. The effects of chronic treatment of these cells with BMPs and of withdrawal following exposure, however, have not been fully elucidated. Thus, the aim of this study was to obtain information about the duration of exposure to recombinant human BMP‐2 (rhBMP‐2) required for expression and retention of osteoblastic characteristics with subsequent formation of a mineralized extracellular matrix in mesenchymal cell cultures. C3H10T1/2 cells and bone marrow stromal cells were cultured with 1 μg/ml rhBMP‐2 for either 0, 7, 14, 21, or 28 days, with the remainder of the 4 week total culture period in the absence of rhBMP‐2. Growth and expression of osteoblastic characteristics were examined at the end of each week. C3H10T1/2 cells responded to increasing duration of exposure to rhBMP‐2 with increased cell growth. Additionally, the longer the cells were exposed to rhBMP‐2, the more fully they expressed and sustained osteoblastic traits, i.e., they exhibited duration of exposure‐dependent higher levels of alkaline phosphatase and osteocalcin and larger total amounts of mineral in the matrix. In comparison, exposure of bone marrow stromal cells to rhBMP‐2 for at least 14 days restrained cell growth and prevented detachment. With respect to osteoblastic traits, stromal cells exposed to rhBMP‐2 also exhibited a dependence on the duration of exposure, however, cultures treated for 14, 21, or 28 days exhibited similar levels of alkaline phosphatase activity and comparable amounts of calcium in the mineralizing matrix. J. Cell. Physiol. 173:93–101, 1997.

Effect of metal ions on the formation and function of osteoclastic cells in vitro

To determine if metal ions play a contributing role in loosening of orthopedic implants, the present work investigated whether sublethal concentrations of ions affect the formation and function of osteoclasts in vitro. Rat bone marrow cells were cultured on slices of devitalized bone and in the presence of ions associated with Co-Cr-Mo and Ti-6A1-4V alloys for up to four weeks. Cultures were assayed for total intracellular protein, used as measure of cell growth, and resorption activity of osteoclastic cells derived from hematopoietic stem cells was quantified using image analysis. Although Co2+ caused delayed toxicity not previously observed during short-term experiments, none of the other ions affected cell proliferation, indicating that the chosen concentrations were sublethal. In general, exposure of bone marrow cultures to ions caused either a decrease or no change in the total area of bone resorption. A decrease in the number of resorption pits formed by osteoclastic cells was primarily responsible for the decrease in total amount of resorption. Therefore, even though cells continued to grow over the entire culture period, less osteoclastic activity was observed. Findings indicate that if metal ions play a role in periprosthetic pathology, they may contribute to implant failure by impairing bone repair while allowing fibrous tissue formation following debris-induced osteolysis.

Biological Interactions on Materials Surfaces

Biological interactions at the tissue/implant material interface can be modulated by surface-linked cell-signalling biological molecules. Collagen type I, the main extracellular matrix protein of bone tissue, has been widely investigated in biomolecular surface modification of bone-contacting titanium implant devices. Literature reports on the biological effects of collagen-based coatings are, however, often contradictory. From a biomolecular surface-engineering perspective, a possible explanation is that the definition “collagen-coated surface” encompasses widely different molecular and supramolecular structures: adsorbed collagen, covalently linked collagen, crosslinked collagen, fibrillar versus monomeric collagen, and many other variation of this theme. Relevant details are not always described and proper surface characterization is often lacking. This chapter attempts to build up a rational frame of reference to describe surface modification of implant devices by collagen type I from a surface chemistry point of view, as well as to discuss relevant implications for process design.