C. Rolfe Howlett

Mechanism of initial attachment of cells derived from human bone to commonly used prosthetic materials during cell culture

The suitability of polymeric biomaterials as surfaces for the attachment and growth of cells has often been investigated in cell culture. In this study the contribution that serum fibronectin (Fn) or vitronectin (Vn) make to the attachment and spreading of cells cultured from explanted human bone (bone-derived cells) during the first 90 min of culture was determined for metallic and ceramic surfaces. The requirement for Fn or Vn for attachment and spreading of bone-derived cells onto stainless steel 316 (SS), titanium (Ti) and alumina (Al2O3) and to polyethyleneterephthalate (PET) was directly tested by selective removal of Fn or Vn from the serum prior to addition to the culture medium. Attachment and spreading of bone-derived cells onto SS, Ti and Al2O3 surfaces were reduced by 73-83% when the cells were seeded in medium containing serum from which the Vn had been removed. Cell attachment and spreading on these surfaces when seeded in medium containing Fn-depleted serum (which contained Vn) were not reduced to the same extent as in the medium containing Vn-depleted serum. The bone-derived cells failed to attach to the surfaces to the same extent when seeded in medium containing serum depleted of both Vn and Fn. Our results show that for human bone-derived cells, the attachment and spreading of cells onto SS, Ti and Al2O3 as well as PET during the first 90 min of a cell culture attachment assay are a function of adsorption of serum Vn onto the surface.

Attachment of human bone cells to tissue culture polystyrene and to unmodified polystyrene: the effect of surface chemistry upon initial cell attachment

Cell culture studies have often been used in the determination of the suitability of biomaterials as surfaces for the attachment and growth of cells. For such studies of surfaces for potential use in bone implants, cells derived from bone may be maintained in culture on tissue culture polystyrene (TCPS). We have determined the contribution that serum fibronectin (FN) or vitronectin (VN) make to the attachment and spreading of cells cultured from explanted human bone (bone-derived cells) during the first 90 min following seeding on culture surfaces. The attachment of bone-derived cells to TCPS was simulated two-fold by the addition of 10% (v/v) fetal bovine serum (FBS) to the seeding culture medium. The roles of FN and VN were determined by selective removal of the FN or VN from the FBS prior to addition to the culture medium. FBS from which the VN had been removed did not have this stimulatory activity. In contrast, the attachment of bone-derived cells onto TCPS from medium containing FN-depleted serum (which contained VN) was the same as when intact FBS was used. There was incomplete attachment of bone-derived cells (27% of cells) when seeded in medium containing FBS depleted of both VN and FN. Our results show that for human bone-derived cells, the attachment onto TCPS of cells planted in medium containing FBS during the first 90 min of culture is principally as a result of adsorption onto the surface of serum VN. As unmodified polystyrene (PS) has also been used previously as a model biomaterial surface, PS was compared to TCPS for attachment of the bone-derived cells. Attachment of bone-derived cells to TCPS was twice that onto PS, both when the medium was serum-free and when it contained FBS. Bone-derived cells attached to TCPS or PS onto which purified VN or FN had been precoated, with VN adsorbed onto PS being as effective as was VN adsorbed onto TCPS. With FN, there was an effect of the polystyrene surface chemistry which was evident in that suboptimal concentrations of FN had a slightly higher potency when adsorbed onto TCPS than did the same concentrations of FN coated onto PS. When preadsorbed onto TCPS, the potency of FN for attachment of bone-derived cells was at least equal to that of VN.

Effect of surface chemical modification of bioceramic on phenotype of human bone-derived cells.

In the search for methods to improve the biocompatibility of prosthetic materials, attention has recently been directed toward the potential use of surface chemical modification and its influence on cellular behavior. This in vitro study investigates the effect of surface chemistry modification of bioceramics on human bone-derived cells (HBDCs) grown on biomaterial surfaces for 2 weeks. Cells were cultured on either alumina (Al2O3), alumina doped with magnesium ions ([Mg]-Al2O3), or hydroxyapatite (HAP), as well as tissue culture polystyrene (TCPS). Expression of alkaline phosphatase (ALP), thrombospondin (Tsp), osteopontin (OP), osteocalcin (OC), osteonectin (ON/SPARC), type I collagen (Col I), and bone sialoprotein (BSP) were determined in terms of mRNAs and proteins. Protein levels for ALP, OP, OC, and BSP were significantly (p < 0. 05) greater at day 5 in HBDCs cultured on [Mg]-Al2O3 compared to those cells grown on Al2O3. At day 14 the levels of ALP, Tsp, Col I, OP, ON/SPARC, and BSP rose significantly (p < 0.05) above those occurring in HBDCs grown on Al2O3, HAP, and TCPS. This suggests that HBDCs from the same patient respond to differences in the surface chemical groups. This study confirms that the chemistry of a substratum, which facilitates cellular adhesion, will enhance cellular differentiation.

Titanium substrata composition influences osteoblastic phenotype: In vitro study.

In spite of observed differences at the interface between boon and either commercially pure titanium [Ti(cpi)] or titanium alloy (Ti-6Al-4V), the mechanism of such a response is ill understood. This prompted further investigation of the influence of similar metals on human bone-derived cells (HBDCs). This study investigated the influence of Ti(cpi) and its alloy on osteoblastic proteins formed by HBDCs grown for 5, 7, 10, and 14 days on these metals and compared them to cells grown on tissue culture polystyrene plates. Messenger RNA and translated proteins that form an array of osteogenic parameters were determined: alkaline phosphatase (ALP), thrombospondin, osteopontin, osteocalcin (OC), osteonectin (ON/SPARC), type I collagen (Col I) and bone sialoprotein (BSP). At the four predetermined time points, cells grown on either Ti(cpi) or Ti-6Al-4V generally expressed similar mRNA levels, while levels of their respective proteins differed. Cells on Ti(cpi) had peak levels for most proteins at day 7, whereas those on Ti-6Al-4V peaked at either day 5 and/or day 7. At day 5 cells grown on Ti-6Al-4V had higher levels of ALP, Col I, ON/SPARC, OC, and BSP than those in Ti(cpi); this difference was not maintained at later time points in culture. The differential regulation of proteins occurring between cells from the same patient grown on titanium and its alloy implies that HBDCs respond to small differences in the surface chemistry and/or microcrystallinity.

A novel technique for quantitative detection of mRNA expression in human bone derived cells cultured on biomaterials

A nonisotopic and quantitative in situ hybridization technique was adapted to investigate the effect of biomaterials on the cellular expression of mRNA from human bone derived cells (HBD cells). HBD cells were cultured for 24 or 48 h on tissue culture plastic, alumina, and ion modified alumina. Osteocalcin, osteopontin, alkaline phosphatase, type I collagen alpha 1, and type I collagen alpha 2 mRNAs were quantified. Protein expression for collagen types I, III, and V, and for anti-human macrophages CD68 (DAKO-CD68, KP1) and CD68 (PG-M1), and anti-human myeloid/histiocyte antigen (DAKO-MAC 387) were determined immunohistochemically using monoclonal antibodies. At 24 and 48 h, levels of mRNA for alkaline phosphatase and osteonectin were greater than mRNA levels for osteopontin, osteocalcin, collagen type I alpha 1, and collagen type I alpha 2 for cells grown on the three substrata. However, at 48 h mRNA levels for alkaline phosphatase and osteonectin were significantly higher on the modified ceramic substrata relative to the native alumina. HBD cells appear to express CD68-KP1 when cultured for 24 h. The techniques provide a sensitive and reproducible assay to evaluate gene and protein expression of cells grown on different substrata.

Effect of ion modification of commonly used orthopedic materials on the attachment of human bone-derived cells

Biomaterials which combine optimum properties of strength and biocompatibility are desirable in improving the long-term performance of implantable medical devices. Our study is aimed at developing technology designed to alter the outer atomic layers of a material to give the desired compatibility with the tissue while retaining the properties of the bulk substratum. Materials used in this study were titanium vanadium alloy (Ti-6Al-4V) and cobalt chromium molybdenum alloy (Co-Cr). Soda lime glass discs and polyethylene terephthalate (PET) acted as controls. A cathode of either Ti-6Al-4V or Co-Cr was used to simultaneously deposit and implant identified substrata. The attachment of human bone-derived cells (HBDC) to various materials was determined using radiolabeling or colorimetric assays. Results show that HBDC adhere preferentially to the unmodified surfaces of Ti-6Al-4V and Ti-6Al-4V on glass compared to the unmodified Co-Cr surfaces and to that of the Co-Cr on glass. Depositing Ti-6Al-4V on Co-Cr gives significantly better attachment of HBDC than when depositing Co-Cr onto Ti-6Al-4V. While cellular attachment to the created surfaces reflects that of the cathodic materials, it is not identical to these materials. Ion deposition/implantation is capable of creating permanent surfaces which reflect the adhesion of source materials not bulk substrata.

Osteogenesis by canine and rabbit bone marrow in diffusion chambers

SummaryOsteogenic activity of canine and rabbit bone marrow and marrow stromal fibroblasts (MSF) derived from marrow culturedin vitro was evaluated using diffusion chambers. Marrow from young dogs and rabbits grown in cell culture produced confluent layers of MSF. Diffusion chambers containing 0.18–7.6×106 allogeneic MSF were inserted into the peritoneal cavities of 5 dogs and 6 rabbits. Chambers recovered from the dogs (15/16) contained only loose fibrous tissue while chambers from rabbits (9/13) contained membranous bone and cartilage. Diffusion chambers implanted with 1.0–32.4×107 fresh allogeneic marrow cells suspended in cell culture medium were inserted into the peritoneal cavities of 11 dogs and 9 rabbits for 3–8 weeks, and after recovery examined histologically. Membranous bone was formed in 4/40 chambers containing canine marrow while bone and hyaline cartilage was formed in 21/27 chambers containing rabbit marrow. This apparent species difference in incidence of bone marrow osteogenesis may relate to a lower concentration of osteogenic precursor cells in canine marrow, a failure of osteogenic precursor cells to differentiate to osteoblasts in a somewhat artificial environmentin vivo (viz diffusion chambers), a lack of cell-matrix interaction to stimulate cell differentiation, inappropriately short diffusion chamber implantation times post grafting, or a difference in ontogenetic stage of development of marrow donors with rabbit cells being physiologically younger.

Anti-inflammatory effects of kappa-opioids in adjuvant arthritis

Current therapies for arthritis are unsatisfactory and cause serious side effects and morbidity. It has been postulated that opioid drugs may block inflammatory mediators and attenuate the joint damage in adjuvant arthritis. However, the importance of opioid receptor subtypes involved in inflammation remains to be determined because data are conflicting in this regard. The present investigation was designed to test the effects of both a kappa-agonist, (+/-)U50488H and a kappa-antagonist, MR2266 on the progression of experimental arthritis. To produce adjuvant arthritis, male Lewis rats were inoculated subcutaneously (s.c.) with 0.05 ml of Freunds complete adjuvant (10 mg/ml) into the right hind paw. The kappa-opioid agonist, (+/-)U50488H (20 mg/kg/d s.c.) and the kappa-opioid antagonist, MR2266 (20 mg/kg/d s.c.) were administered for 3 days during the primary inflammatory phase of adjuvant arthritis. There were four treatment groups; group I were non-arthritic controls and received paraffin oil vehicle and opioid injections; group II were arthritic controls and received adjuvant and saline injections; group III received adjuvant and agonist and group IV received adjuvant and antagonist. The progression of adjuvant arthritis from day 0 to 24 was monitored by body weight change, hind limb size (ipsilateral and contralateral) and a total severity score for each clinical observation of gait, coat and limb condition. On day 24 histology and radiography of the contralateral limb was performed. There was less soft-tissue swelling, as judged by time-averaged % change in the volume of the contralateral limb, in both agonist (mean +/- se: 82 +/- 5) and antagonist (77 +/- 4) treated rats compared to untreated arthritic controls (99 +/- 5, p < 0.05). Other clinical measures of severity were not different between untreated and opioid-treated arthritic rats. However, the joint damage as judged by radiography was lower in kappa agonist treated rats (2.6 +/- 0.5, p < 0.05) compared to untreated controls (4.1 +/- 0.5) and antagonist treatment (4.4 +/- 0.5). Microscopic pathological scores were also significantly lower in agonist (2.8 +/- 0.3, p < 0.05) compared to both antagonist treated rats (4.2 +/- 0.1) and vehicle-treated controls (3.6 +/- 0.2). The results of this study show that kappa-opioid receptor agonists but not antagonists attenuate the progression of experimental arthritis. These observations have important implications for the evaluation and use of kappa-opioid agents in the management of arthritis.

Characteristles of Advanced Porous and Textured Surface Pacemaker Electrodes

Reduced stimulation thresholds, improved sensing and betler attachment have been claimed for totally porous and porous surfaced electrodes. In this study, the potential for clinical use of two new types of porous electrodes and a non‐porous, textured high microsurface area electrode, has been evaluated by comparison with equivalenf sized, smooth non‐porous controls. Eighteen sintered and seven laser drilled porous electrodes, seven non‐porous textured electrodes, and sixteen controls, were implanted singly in fhe right ventricles of sheep. Measurements of threshold, pacing, sensing and bulk impedances were taken at regular intervals for up to 180 days. At sacrifice, only three of the thirteen non‐porous confrols were attached. AU laser porous electrodes, apart from two which were dislodged, were attached, as were eieven of fifteen sintered porous, and five of seven textured non‐porous electrodes. Tissue ingrowth was found for both porous electrode types. Stimulation threshoJds were not statistically different for all electrode types (p < 0.05). Pacing and bulk impedances of the two porous and surface textured electrodes were significantly higher (p < 0.10, p < 0.05, respectively) than those of controls. The three new electrodes exhibited similar chronic sensing impedance vaJues, 30% less than equivalent non‐porous electrodes. The similar sensing performance of the porous and high microsurface area non‐porous electrodes indicates that the nature of the external surface, rather than internal porosity, determines sensing impedance. All three new electrode types showed improved attachment and sensing compared with similar smooth eJectrodes. The laser porous electrode, which permits fixation by tissue ingrowth and maintainssimplicity of construction, is promising for routine clinical use.

Quantitative aspects of anin situ hybridization procedure for detecting mRNAs in cells using 96-well microplates

The universal quantitation of the DNA hybridization reaction has been a goal sought by many researchers. Part of this search has been the need to develop a rapid, sensitive, easy-to-perform, and quantitative method to measure the abundance of specific mRNAs directly within cells. Conventionally mRNA detection can be done by advanced quantitativein situ hybridization (ISH) using either image analysis or fluorescencein situ hybridization (FISH), or indirectly by extraction of mRNA from cells or tissue and using Northern blot or quantitative polymerase chain reaction (PCR). We examined the quantitative nature of probe binding to intracellular mRNA in a sensitive and easy-to-use nonisotopic method of ISH previously developed in our laboratories. The method is applicable to isolated primary cells or cells in culture. The procedural details are very simple, with cells being centrifuged into 96-well microplates, fixed with formalin, and pretreated with Triton X-100 and Nonidet P-40 before photobiotin-labeled cDNA probes are applied. Biotin from the hybridization of probe to target is detected using multiple applications of streptavidin and biotinylated alkaline phosphatase and visualized by thep-nitrophenyl phosphate conversion method. The quantitative parameters of the ISH procedure were determined by measuring the levels of expression of erythropoietin (EPO) mRNA and its translated protein in transfected COS-7 cells. There is a log-linear relationship between the levels of signal obtained in the ISH reaction in 96-well microplates and the EPO protein levels measured by enzyme-linked immunosorbent assay (ELISA). This demonstrated relationship is important in the standardization and use of these procedures to measure quantitatively mRNAs within cells.