B. D. Boyan

Titanium surface roughness alters responsiveness of MG63 osteoblast‐like cells to 1α,25‐(OH)2D3

Surface roughness has been shown to affect dif- ferentiation and local factor production of MG63 osteoblast- like cells. This study examined whether surface roughness alters cellular response to circulating hormones such as 1a,25-(OH)2D3. Unalloyed titanium (Ti) disks were pre- treated with HF/HNO3 (PT) and then were machined and acid-etched (MA). Ti disks also were sandblasted (SB), sand- blasted and acid etched (CA), or plasma sprayed with Ti particles (PS). The surfaces, from smoothest to roughest, were: PT, MA, CA, SB, and PS. MG63 cells were cultured to confluence on standard tissue culture polystyrene (plastic) or the Ti surfaces and then treated for 24 h with either 10 ˛8 M or 10 ˛7 M 1a,25-(OH)2D3 or vehicle (control). Cellular re- sponse was measured by assaying cell number, cell layer alkaline phosphatase specific-activity, and the production of osteocalcin, latent (L) TGFb, and PGE2. Alkaline phospha- tase activity was affected by surface roughness; as the sur- face became rougher, the cells showed a significant increase in alkaline phosphatase activity. Addition of 1a,25-(OH)2D3 to the cultures caused a dose-dependent stimulation of al- kaline phosphatase activity that was synergistic with the

The Role of Implant Surface Characteristics in the Healing of Bone

The surface of an implant determines its ultimate ability to integrate into the surrounding tissue. The composite effect of surface energy, composition, roughness, and topography plays a major role during the initial phases of the biological response to the implant, such as protein adsorption and cellular adherence, as well as during the later and more chronic phases of the response. For bone, the successful incorporation (and hence rigid fixation) of an alloplastic material within the surrounding bony bed is called osteointegration. The exact surface characteristics necessary for optimal osteointegration, however, remain to be elucidated. This review will focus on how surface characteristics, such as composition and roughness, affect cellular response to an implant material. Data from two different culture systems suggest that these characteristics play a significant role in the recruitment and maturation of cells along relevant differentiation pathways. In the case of osteointegration, if the implant surface is inappropriate or less than optimal, cells will be unable to produce the appropriate complement of autocrine and paracrine factors required for adequate stimulation of osteogenesis at the implant site. In contrast, if the surface is appropriate, cells at the implant surface will stimulate interactions between cells at the surface and those in distal tissues. This, in turn, will initiate a timely sequence of events which include cell proliferation, differentiation, matrix synthesis, and local factor production, thereby resulting in the successful incorporation of the implant into the surrounding bony tissue.

Effect of titanium surface roughness on chondrocyte proliferation, matrix production, and differentiation depends on the state of cell maturation

Although it is well accepted that implant success is dependent on various surface properties, little is known about the effect of surface roughness on cell metabolism or differentiation, or whether the effects vary with the maturational state of the cells interacting with the implant. In the current study, we examined the effect of titanium (Ti) surface roughness on chondrocyte proliferation, differentiation, and matrix synthesis using cells derived from known stages of endochondral development. Chondrocytes derived from the resting zone (RCs) and growth zone (GCs) of rat costochondral cartilage were cultured on Ti disks that were prepared as follows: HF-HNO3-treated and washed (PT); PT-treated and electropolished (EP); fine sand-blasted, HCl-H2SO4-etched, and washed (FA); coarse sand-blasted, HCl-H2SO4-etched, and washed (CA); or Ti plasma-sprayed (TPS). Based on surface analysis, the Ti surfaces were ranked from smoothest to roughest: EP, PT, FA, CA, and TPS. Cell proliferation was assessed by cell number and [3H]-thymidine incorporation, and RNA synthesis was assessed by [3H]-uridine incorporation. Differentiation was determined by alkaline phosphatase specific activity (AL-Pase). Matrix production was measured by [3H]-proline incorporation into collagenase-digestible (CDP) and noncollagenase-digestible (NCP) protein and by [35S]-sulfate incorporation into proteoglycan. GCs required two trypsinizations for complete removal from the culture disks; the number of cells released by the first trypsinization was generally decreased with increasing surface roughness while that released by the second trypsinization was increased. In RC cultures, cell number was similarly decreased on the rougher surfaces; only minimal numbers of RCs were released by a second trypsinization. [3H]-thymidine incorporation by RCs decreased with increasing surface roughness while that by GCs was increased. [3H]-Uridine incorporation by both GCs and RCs was greater on rough surfaces. Conversely, ALPase in the cell layer and isolated cells of both cell types was significantly decreased. GC CDP and NCP production was significantly decreased on rough surfaces while CDP production by RC cells was significantly decreased on smooth surfaces. [35S]-sulfate incorporation by RCs and GCs was decreased on all surfaces compared to tissue culture plastic. The results of this study indicate that surface roughness affects chondrocyte proliferation, differentiation, and matrix synthesis, and that this regulation is cell maturation dependent.

17β-estradiol-BSA conjugates and 17β-estradiol regulate growth plate chondrocytes by common membrane associated mechanisms involving PKC dependent and independent signal transduction

Nuclear receptors for 17β‐estradiol (E2) are present in growth plate chondrocytes from both male and female rats and regulation of chondrocytes through these receptors has been studied for many years; however, recent studies indicate that an alternative pathway involving a membrane receptor may also be involved in the cell response. E2 was found to directly affect the fluidity of chondrocyte membranes derived from female, but not male, rats. In addition, E2 activates protein kinase C (PKC) in a nongenomic manner in female cells, and chelerythrine, a specific inhibitor of PKC, inhibits E2‐dependent alkaline phosphatase activity and proteoglycan sulfation in these cells, indicating PKC is involved in the signal transduction mechanism. The aims of the present study were: (1) to examine the effect of a cell membrane‐impermeable 17β‐estradiol‐bovine serum albumin conjugate (E2‐BSA) on chondrocyte proliferation, differentiation, and matrix synthesis; (2) to determine the pathway that mediates the membrane effect of E2‐BSA on PKC; and (3) to compare the action of E2‐BSA to that of E2. Confluent, fourth passage resting zone (RC) and growth zone (GC) chondrocytes from female rat costochondral cartilage were treated with 10−9 to 10−7 M E2 or E2‐BSA and changes in alkaline phosphatase specific activity, proteoglycan sulfation, and [3H]‐thymidine incorporation measured. To examine the pathway of PKC activation, chondrocyte cultures were treated with E2‐BSA in the presence or absence of GDPβS (inhibitor of G‐proteins), GTPγS (activator of G‐proteins), U73122 or D609 (inhibitors of phospholipase C [PLC]), wortmannin (inhibitor of phospholipase D [PLD]) or LY294002 (inhibitor of phosphatidylinositol 3‐kinase). E2‐BSA mimicked the effects of E2 on alkaline phosphatase specific activity and proteoglycan sulfation, causing dose‐dependent increases in both RC and GC cell cultures. Both forms of estradiol inhibited [3H]‐thymidine incorporation, and the effect was dose‐dependent. E2‐BSA caused time‐dependent increases in PKC in RC and GC cells; effects were observed within three minutes in RC cells and within one minute in GC cells. Response to E2 was more robust in RC cells, whereas in GC cells, E2 and E2‐BSA caused a comparable increase in PKC. GDPβS inhibited the activation of PKC in E2‐BSA‐stimulated RC and GC cells. GTPγS increased PKC in E2‐BSA‐stimulated GC cells, but had no effect in E2‐BSA‐stimulated RC cells. The phosphatidylinositol‐specific PLC inhibitor U73122 blocked E2‐BSA‐stimulated PKC activity in both RC and GC cells, whereas the phosphatidylcholine‐specific PLC inhibitor D609 had no effect. Neither the PLD inhibitor wortmannin nor the phosphatidylinositol 3‐kinase inhibitor LY294022 had any effect on E2‐BSA‐stimulated PKC activity in either RC or GC cells. The classical estrogen receptor antagonist ICI 182780 was unable to block the stimulatory effect of E2‐BSA on PKC. Moreover, the classical receptor agonist diethylstilbestrol (DES) had no effect on PKC, nor did it alter the stimulatory effect of E2‐BSA. The specificity of the membrane response to E2 was also demonstrated by showing that the membrane receptor for 1α,25‐(OH)2D3 was not involved. These data indicate that the rapid nongenomic effect of E2‐BSA on PKC activity in RC and GC cells is dependent on G‐protein‐coupled PLC and support the hypothesis that many of the effects of E2 involve membrane‐associated mechanisms independent of classical estrogen receptors. J. Cell. Biochem. 81:413–429, 2001.

Growth plate chondrocytes store latent transforming growth factor (TGF)- β1 in their matrix through latent TGF-β1 binding protein-1

Osteoblasts produce a 100 kDa soluble form of latent transforming growth factor beta (TGF‐β) as well as a 290 kDa form containing latent TGF‐β binding protein‐1 (LTBP1), which targets the latent complex to the matrix for storage. The nature of the soluble and stored forms of latent TGF‐β in chondrocytes, however, is not known. In the present study, resting zone and growth zone chondrocytes from rat costochondral cartilage were cultured to fourth passage and then examined for the presence of mRNA coding for LTBP1 protein. In addition, the matrix and media were examined for LTBP1 protein and latent TGF‐β. Northern blots, RT‐PCR, and in situ hybridization showed that growth zone cells expressed higher levels of LTBP1 mRNA in vitro than resting zone cells. Immunohistochemical staining for LTBP1 revealed fine fibrillar structures around the cells and in the cell matrix. When the extracellular matrix of these cultures was digested with plasmin, LTBP1 was released, as determined by immunoprecipitation. Both active and latent TGF‐β1 were found in these digests by TGF‐β1 ELISA and Western blotting. Immunoprecipitation demonstrated that the cells also secrete LTBP1 which is not associated with latent TGF‐β, in addition to LTBP1 that is associated with the 100 kDa latent TGF‐β complex. These studies show for the first time that latent TGF‐β is present in the matrix of costochondral chondrocytes and that LTBP1 is responsible for storage of this complex in the matrix. The data suggest that chondrocytes are able to regulate both the temporal and spatial activation of latent TGF‐β, even at sites distant from the cell, in a relatively avascular environment. J. Cell. Physiol. 177:343–354, 1998.

1,25(OH)2D3 Regulates Protein Kinase C Activity Through Two Phospholipid-Dependent Pathways Involving Phospholipase A2 and Phospholipase C in Growth Zone Chondrocytes

We have previously shown that 1,25‐dihydroxyvitamin D3 (1,25(OH)2D3) plays a major role in growth zone chondrocyte (GC) differentiation and that this effect is mediated by protein kinase C (PKC). The aim of the present study was to identify the signal transduction pathway used by 1,25(OH)2D3 to stimulate PKC activation. Confluent, fourth passage GC cells from costochondral cartilage were used to evaluate the mechanism of PKC activation. Treatment of GC cultures with 1,25(OH)2D3 elicited a dose‐dependent increase in both inositol‐1,4,5‐trisphosphate and diacylglycerol (DAG) production, suggesting a role for phospholipase C and potentially for phospholipase D. Addition of dioctanoylglycerol to plasma membranes isolated from GCs increased PKC activity. Neither pertussis toxin nor choleratoxin had an inhibitory effect on PKC activity in control or 1,25(OH)2D3‐treated GCs, indicating that neither Gi nor Gs proteins were involved. Phospholipase A2 inhibitors, quinacrine, OEPC (selective for secretory phospholipase A2), and AACOCF3 (selective for cytosolic phospholipase A2), and the cyclooxygenase inhibitor indomethacin decreased PKC activity, while the phospholipase A2 activators melittin and mastoparan increased PKC activity in GC cultures. Arachidonic acid and prostaglandin E2, two downstream products of phospholipase A2 action, also increased PKC activity. These results indicate that 1,25(OH)2D3‐dependent stimulation of PKC activity is regulated by two distinct phospholipase‐dependent mechanisms: production of DAG, primarily via phospholipase C and production of arachidonic acid via phospholipase A2.

24,25-(OH)2D3 regulates cartilage and bone via autocrine and endocrine mechanisms

The purpose of this paper is to summarize recent advances in our understanding of the physiological role of 24(R),25(OH)(2)D(3) in bone and cartilage and its mechanism of action. With the identification of a target cell, the growth plate resting zone (RC) chondrocyte, we have been able to use cell biology methodology to investigate specific functions of 24(R),25(OH)(2)D(3) and to determine how 24(R),25(OH)(2)D(3) elicits its effects. These studies indicate that there are specific membrane-associated signal transduction pathways that mediate both rapid, nongenomic and genomic responses of RC cells to 24(R),25(OH)(2)D(3). 24(R),25(OH)(2)D(3) binds RC chondrocyte membranes with high specificity, resulting in an increase in protein kinase C (PKC) activity. The effect is stereospecific; 24R,25(OH)(2)D(3), but not 24S,25-(OH)(2)D(3), causes the increase, indicating a receptor-mediated response. Phospholipase D-2 (PLD2) activity is increased, resulting in increased production of diacylglycerol (DAG), which in turn activates PKC. 24(R),25(OH)(2)D(3) does not cause translocation of PKC to the plasma membrane, but activates existing PKCalpha. There is a rapid decrease in Ca(2+) efflux, and influx is stimulated. 24(R),25(OH)(2)D(3) also reduces arachidonic acid release by decreasing phospholipase A(2) (PLA(2)) activity, thereby decreasing available substrate for prostaglandin production via the action of cyclooxygenase-1. PGE(2) that is produced acts on the EP1 and EP2 receptors expressed by RC cells to downregulate PKC via protein kinase A, but the reduction in PGE(2) decreases this negative feedback mechanism. Both pathways converge on MAP kinase, leading to new gene expression. One consequence of this is production of new matrix vesicles containing PKCalpha and PKCzeta and an increase in PKC activity. The chondrocytes also produce 24(R),25(OH)(2)D(3), and the secreted metabolite acts directly on the matrix vesicle membrane. Only PKCzeta is directly affected by 24(R),25(OH)(2)D(3) in the matrix vesicles, and activity of this isoform is inhibited. This effect may be involved in the control of matrix maturation and turnover. 24(R),25(OH)(2)D(3) causes RC cells to mature along the endochondral developmental pathway, where they become responsive to 1alpha,25(OH)(2)D(3) and lose responsiveness to 24(R),25(OH)(2)D(3), a characteristic of more mature growth zone (GC) chondrocytes. 1alpha,25(OH)(2)D(3) elicits its effects on GC through different signal transduction pathways than those used by 24(R),25(OH)(2)D(3). These studies indicate that 24(R),25(OH)(2)D(3) plays an important role in endochondral ossification by regulating less mature chondrocytes and promoting their maturation in the endochondral lineage.

The Titanium-Bone Cell Interface In Vitro: The Role of the Surface in Promoting Osteointegration

One of the major advances in the use of metals for long-term implants was the serendipitous use of titanium (Ti). The nonreactive properties of this metal [1] made it an ideal material for the aerospace industry. Many of these same properties made it equally ideal for use in the body. Unlike stainless steel, Ti forms a surface oxide that prevents leaching of ions [2]. The oxide is biocompatible in that there is little, if any, immune response [3]. Early literature noted the lack of an immune response and defined Ti as biologically inert [4]. While we now know that Ti is not inert, there is no question that it is exceptionally well-tolerated by the body. Consequently, Ti has become the material of choice for dental implants [5] (See Chaps. 24 and 25) and cardiovascular stents [6] (See Chap. 26), and its alloys are commonly used in orthopaedics where greater strength is needed [7] (See Chap. 21).

Evidence for receptors specific for 17βestradiol and testosterone in chondrocyte cultures

Recently, sex hormones were shown to stimulate chondrocyte differentiation and matrix protein synthesis in vitro in a sex-specific and maturation-dependent manner. The aim of the present study was to determine whether cytosolic receptors in these cells would specifically bind 17 beta-estradiol and testosterone, and if so, whether binding was gender- and maturation-dependent. Confluent, fourth passage cultures of cells derived from male or female rat costochondral growth zone and resting zone cartilage were homogenized and specific binding of 17 beta-estradiol or testosterone measured in the cytosolic fraction. Scatchard analysis indicated the presence of a high-affinity 17 beta-estradiol receptor (Kd = 4.5 to 8.7 x 10(-11) M), with low binding capacity (3.9 to 11.2 fmol/mg protein). Chondrocytes from female rats were found to have a significantly greater binding capacity for 17 beta-estradiol than chondrocytes from male rats. However, cells from both sexes had binding capacities that were independent of cell maturation. A high-affinity testosterone receptor (Kd = 4.3 to 6.3 x 10(-11) M) with low binding capacity (4.1 to 5.9 fmol/mg protein) was found in both males and females, but no difference in binding capacity was noted, either as a function of gender or stage of cell maturation. Immunohistochemistry using antibodies against 17 beta-estradiol and testosterone and the 17 beta-estradiol nuclear receptor (D-75) confirmed that 17 beta-estradiol and testosterone receptors were present in chondrocytes from both male and female rats. These data demonstrate that chondrocytes from growth zone and resting zone cartilage are capable of binding both 17 beta-estradiol and testosterone. This suggests that these hormones mediate their direct effects on chondrocytes via receptors specific for their appropriate ligand. The sex-specific effects of 17 beta-estradiol may be due to differences in receptor number between chondrocytes derived from female and male rats. In contrast, the sex-specific effects of testosterone may be regulated at the post receptor level since no differences in binding capacity were found between males and females.

17β-Estradiol regulation of protein kinase C activity in chondrocytes is sex-dependent and involves nongenomic mechanisms

17β‐Estradiol (E2) regulates growth plate chondrocyte differentiation in both a sex‐ and cell maturation–dependent manner, and the sex‐specific effects of E2 appear to be mediated in part by membrane events. In this study, we examined whether E2 regulates protein kinase C (PKC) in a cell‐maturation and sex‐specific manner and whether E2 uses a nongenomic mechanism in regulating this enzyme. In addition, we determined if PKC mediates the E2‐dependent stimulation of alkaline phosphatase activity seen in chondrocytes. Confluent, fourth passage resting zone (RC) and growth zone (GC) chondrocytes from male and female rat costochondral cartilage were treated with 10−10 to 10−7 M E2. E2 caused a dose‐dependent increase in PKC in RC and GC cells from female rats. Peak stimulation was at 90 min. Increased PKC was evident by 3 min in both RC and GC and was still evident in RC cells at 720 min, but in GC cells activity returned to baseline by 270 min. Actinomycin D had no effect at 9, 90, 270, or 720 min, but there was a small decrease in E2‐stimulated PKC in RC treated with cycloheximide at 90 and 270 min and in GC treated for 90 min. E2 increased cytosolic and membrane PKC at 9 min and by 90 min promoted translocation of PKC activity from the cytosol to the membranous compartment of female RC cells. Antibodies specific for the α, β, δ, ε, and ζ isoforms of PKC revealed that PKCα in female GC and RC cells is activated by E2. There was a small, but statistically significant, increase in PKC in male RC cells in response to E2, but it was not dose‐dependent, and no effect of E2 was noted in male GC cells. 17α‐estradiol, an inactive isomer of E2, did not affect PKC specific activity in RC or GC cells from either female or male rats. Chelerythrine, a specific inhibitor of PKC, inhibited E2‐dependent alkaline phosphatase activity, indicating that E2 mediates its rapid effects on alkaline phosphatase via PKC. J. Cell. Physiol. 176:435–444, 1998.