RESPONSE…
Bioactivity of Osteoblasts to Wear Debris Generated From Orthopedic Devices
One of the major modes of orthopedic implant failure is aseptic loosening. Many researchers have postulated that the main reason behind the immunoresponse causing the aseptic loosening of orthopedic implants is the presence of the small micro-particles generated from the articulation of load bearing surfaces around the surrounding tissue of the implant. These microparticles, appropriately referred to as wear or particle debris have been shown to be phagocytosed by macrophages. The phagocytosis of the microparticles produces a localized foreign body response and a wave of cellular events involving osteoclast activation, which leads to bone resorption, and eventually to the removal of the implant. Since it is has been postulated that the foreign body reaction to the microparticles is mediated by the osteoblastic response, this section will investigate the effects of particle wear debris on several parameters of osteoblast activity. The approach will focus on the functionality of osteoblasts through measurements of osteocalcin production, collagen synthesis and proliferative indexes after exposure to simulated wear debris of polyethylene, polymethylmetacrylate (PMMA) and titanium.
The success of an implant is dependent on the properties of the biomaterials and their associated applications. For instance, in most applications, metals are primarily used in the load bearing joint replacement while the articulating surfaces of the joints typically consist of ultra dense polymers (Vrouwenvelde). Despite the application, however, particulate wear debris is a shared conundrum in all forms of biomaterials used in orthopedic implants. The ill effects of wear debris have been detected in polymeric based implant materials such as polyethylene and polystyrene as well as metallic based implant materials such as titanium and cobalt chromium in addition to cements such as polymethylmetacrylate (PMMA) or hydroxyapatite. The polymeric particulates come from mechanical friction while the metallic particulates arise from chemical breakdown and interactions with the local biological system and metallic ions. Both types of foreign particles generally produce the same inflammatory response, which may lead to bone resorption, osteolysis, loosening, or fracture of the bone.
Of the gamut of particulates producing deleterious effects surrounding local tissue, polyethylene wear debris may be the single most nocuous. It's produced by friction between the polyethylene socket and the head of a femoral component in a hip prosthesis joint or between the femoral and tibial components of knee joint replacements.
In order to truly characterize the osteoblastic reactivity to these wear particles a series of experiments were performed to realistically model the interactions of osteoblasts when introduced to the wear debris. The osteoblastic response to polyethylene wear debris was experimentally achieved through the addition of sterilized 1 m m diameter ultrahigh molecular weight polyethylene microparticles to confluent monolayers of MG63 osteoblast-like cells (Dean). Scanning Electron Microscopy of the ultra high molecular weight polyethylene particles indicated that the majority of the spheres were round or spherical with a rough modular surface containing numerous pits and small spherulites. The spherulites, which ranged from less than 1 m m in diameter to 6 m m, were either physically attached to larger particles or were resting on the surface of the large particles. 90% of the larger polyethylene particles used in the experiments ranged between 40 m m to 180 m m in diameter. Morphological assessment indicated that the microparticles used in the experiments are also comparable in size to those found in periprosthetic tissue gathered from human hip resections (Dean). Experiments to depict the biological response of osteoblasts to polyethylene wear debris included osteocalcin production and proliferative examination.
PMMA is commonly used as the fixing agent for the stem component for total hip arthroplasty. PMMA wear debris is produced from the friction and fragmentation of the cement mantle surrounding the prosthesis (Zambonin). Though the interaction of cement and surrounding bone tissue has been well characterized , very little information exists regarding osteoblasts and their reactivity to the PMMA microparticles.
To characterize the physiologic interface of cemented joint arthroplasty, human osteoblasts were exposed to PMMA particles. The microparticles which had a size distribution of .5 m m to 25 m m, were produced from a steel mortar and sterilized with light (Zambonin). They were added to osteoblasts derived from bone fragments generated after 1 hour of total hip arthroplasty in cell culture dishes. Slews of experiments were performed to determine the interactions between osteoblasts and cement wear debris.
Similar to the polymers and cements, metallic implants have demonstrated debris generation despite their mechanical stability. Of this particular class of biomaterials, Stainless steel and cobalt- chromium alloys reportedly display slightly higher accounts of metallic wear and corrosion as compared to titanium alloys. Since titanium alloys have excellent mechanical properties and good biotolerancy, they have been used in a host of surgical applications including, the gamut of join replacements (total hip, knee, ankle, shoulder and finger) as well as implantable screws, plates and wire. They are unequivocally regarded as one of the most biocompatible metals available today. Despite their popularity, titanium alloys are not free from producing harmful particulate wear debris as other implant materials. In fact, they generate similar if not identical inflammatory response like other particulate matter in close contact with bone. Metallic particulates are produced from the simple corrosion of the metal in the body, corrosion resulting from the mismatch of implant alloys or overloading of the implant as often seen in titanium alloy wire (Zambonin). The associated particle sizes of debris resulting from the corrosion of implanted titanium are typically in the range of 3 microns or less (Yao).
In the experiments to determine osteoblast viability in the presence of titanium wear debris, commercially pure titanium particles, on the order of 1 to 3 m m were sterilized by irradiation and added to MG-63 osteoblast like cells. Collagen synthesis and proliferative index studies were performed to analyze the effects of titanium wear debris to cultured osteoblasts.
Since osteoblasts represent the bone-generating cell, many efforts have been directed towards the reactivity of osteoblasts to inorganic materials. The role played by macrophages and the entire immune response has been thoroughly examined but the data concerning the interactions of osteoblasts and wear debris are in short supply. Despite the extreme importance of characterizing and mediating the deleterious effect of wear debris to osteoblasts the best method of approach to solve this problem has not been resolved (Martinez).
In order for the results of the appropriate experiments to be comparatively analyzed, the heterogeneity among bone cell populations must first be acknowledged. With that said, there are various quantifiable osteoblast markers which can be analyzed between the slightly varying populations. Some of these include histological and biochemical parameters such as cell morphology, proliferation and differentiation, collagen production, alkaline phosphatase activity and osteocalcin production, however, this report will investigate the effects of microparticle wear debris on osteocalcin production, collagen synthesis and proliferation of osteoblasts.
One of the most utilized techniques to determine osteoblast functionality and health is measurement of collagen synthesis. Collagen, which is directly produced from osteoblasts, makes up the extracellular organic matrix of bone. The matrix has a rubbery property and is responsible for the tensile strength of bone (Sherwood). Hence, measuring the production of collagen production directly measures the vigor, health, and functionality of osteoblasts. In the experiments performed to test osteoblast functionality in the presence of PMMA and titanium microparticles, collagen synthesis of target osteoblasts were measured.
It has been established that increases in osteocalcin levels around osteoblasts are directly linked to increased osteoblastic differentiation and mineralization of the bone matrix. The increased differentiation and mineralization leads to the calcified bony matrix formation. Hence, osteocalcin production serves as an important marker for assessing osteoblast functionality. Researchers used this marker to determine the effect of PMMA and Polyethylene wear debris on human derived osteoblasts and human osteoblast-like cells MG-63, respectively. Osteocalcin levels were determined with a radioimmunoassay after the osteoblasts were exposed to PMMA and Polyethylene microparticles (Zambonin, Dean).
The clearest way to determine osteoblast viability is by determining cell proliferation. All of the researchers which studied the effects of wear debris used this technique to gauge overall osteoblast health and vigor. For the osteoblasts exposed to titanium and PMMA particles, proliferation was determined by incorporation of H-thymidine with a microplate assay system (Yao, Zambonin) while the studies using Polyethylene particles used a Coulter counter to asses
s proliferation.
Polymethylmetacrylate (PMMA)
Osteoblasts cultured with PMMA particles demonstrated reduced collagen synthesis shown by lower levels of H-proline incorporation by the osteoblasts. The significant inhibition was 63% of the untreated control osteoblasts. (fig 2., Zambonin). In the graph depicted in the figure below, the collagen synthesis of the osteoblasts to PMMA particles is expressed as percent of control.
The osteoblasts exposed to PMMA particles significantly increased their production of osteocalcin.
Osteoblast proliferation rate was significantly inhibited as expressed by reduced the H-thymidine uptake after exposure to PMMA particles. The results in (fig. 1, Zambonin, p.363) indicated that the viability was 58% that of untreated control cells. The results were recreated to express percent H-thymidine uptake as a percent of control.
Total collagen synthesis in the human osteoblast-like cells MG-63 that were treated with titanium particles faced a similar decrease in collagen production. Collagen synthesis in these cells significantly decreased by 46% in culture medium and by 68% in cell lysates versus that of untreated controls. Both Collage type-1 and type-2 were significantly suppressed. (fig 3., Yao). The graph of collagen synthesis by osteoblasts among titanium particles was recreated as percent of control so differences could be compared to that of the osteoblasts exposed to PMMA particles.
Osteoblast proliferation varied depending on the composition and dosage of microparticles. The osteoblasts, which were loaded with titanium particles, did not have any changes in viability. After 72 hours of incubation, the microparticles had no effect on the incorporation of H-thymidine as compared to that of untreated control cells. The osteoblasts that were exposed to titanium particles had no significant differences in proliferation when compared to when compared to untreated controls (Yao).
The study indicated that ultra high molecular weight polyethylene had no significant effect on osteocalcin production by MG-63 osteoblast-like cells exposed to 0.1 mg/ml dilutions from 1:10,000 to 1:10.
Exposures to polyethylene microparticles resulted in a statistically significant increase in osteoblast proliferation and thus increase in H-thymidine incorporation, with or without the use of inserts. Inserts were used to keep the microparticles from coming into contact with the osteoblasts. Such a technique allowed the researchers to test if any leachable effect from the microparticles were causing harmful effects to the cells. The net effect of on osteoblast proliferation was proven to be dose dependent. Significant differences were observed at 0.1 mg/ml polyethylene microparticle suspension dilutions of 1:1,000, 1:100, and 1:10. The osteoblasts that were exposed to the 1:10 dilution of particles increased 30%. The number of osteoblasts that were seeded directly in the wells was approximately twice that of osteoblasts which were seeded in inserts though both populations significantly increased. Since other experiments near constant phenotypic expression (refer to osteocalcin results), the increase in proliferation of osteoblasts in the presence of polyethylene microparticles may indicate a shift in the cell cycle (Dean). It is not known whether the increased proliferation is due to the preferential growth of a subset of the cells present or if it is due to a transient shift that will revert once the particles are removed. The results do indicate that there is a change to a less differentiated phenotype since no increases in osteocalcin production was observed. Although proliferation was markedly increased with polyethylene, other researchers have reported osteoblast proliferation was unaffected by the presence of polystyrene and other polymers.
Summary
Though the osteoblasts used in the three studies of bioresponse to wear debris come from different stocks, each of the studies’ control populations expressed normal values for various osteoblast markers. Hence, we can reasonably compare the results of wear particles on the basis of percent of control. Human osteoblast-like cells MG-63 have been shown to not form mineralized matrix. Thus, the reduced collagen production as seen in the MG-63 cells exposed to Titanium is not surprising. However, this effect on overall osteoblast reactivity to the wear particle is marginalized since other parameters were measured to assess osteoblast reactivity.
In cell culture, it has been established that the size of particles may alter the surface structure of substrates which the cells adhere. The alterations in the surface structure could have an effect on the phenotypic expressions of the cells and thereby drastically affecting the cells. Studies have reported that particle sizes larger than 3 m m have no effect on osteoblasts because the particles cannot be ingested. However, smaller particles that do get ingested may occlude access to nutrients by cells. Cell membranes may also get damaged in the attempts to endocytose these particles. Since all particles sizes used in the three different studies were relatively small and comparable to those collected from periprosthetic tissues, a reasonable conclusion can be made that suggests the bioactivity of the osteoblasts are due to the ingestion of the wear debris.
The polyethylene particles used in the investigation were approximately 1 m m in diameter, the titanium particles were less than the 3 m m and the PMMA powder used in the experiments ranged in diameter between 0.5 and 25 m . Researchers have also shown that bone formation in vivo can be markedly affected by the presence of materials that are not even in direct contact with the cells. Hence, it was not surprising that researchers found increased proliferation in osteoblasts that were in direct contact with polyethylene particles than those that were not.
The length of time the osteoblasts were treated with particles has an affect of the results seen. The longer the cells are allowed to culture with the spheres the more definitive changes can be observed from the marker. In in vitro studies, it can be reasonably asserted that the longer the cells stay in culture with microparticles, the slower their growth. Growth is dependent on the size of the culture dish. The smaller sized culture dishes would tend to inhibit osteoblast proliferation sooner than a larger sized dish would.