Biomaterials in orthopedic surgery: effects of a nickel-reduced stainless steel on in vitro proliferation and activation of human osteoblasts

A new austenitic stainless steel compound, P558, has been widely recognized to have good mechanical properties, excellent potential for corrosion resistance and negligible nickel ion release, making it a promising substitute for more expensive metallic prostheses with limited machinable features. The effect of P558 was studied in vitro and human osteoblast- like cells (MG63) were cultured directly on P558, Ti6Al4V alloy (Ti), and polystyrene (Control) for 72 hours. Osteoblast functions were evaluated by assaying cell proliferation and synthetic activity after 1.25(OH)2D3 stimulation. Results demonstrated that growth of MG63 on P558 was not negatively affected when compared to the Ti and Control groups and showed no alteration in the production of ALP, NO and PICP. Moreover, IL-6 was lower, whereas OC and TGFbeta1 were significantly higher. SEM images revealed that cells proliferated and differentiated on P558 without any alteration in their morphology. The current findings have demonstrated that P558 promotes osteoblast proliferation, activation and differentiation without negative effects and, thus, its good biocompatibility when used for orthopedic application.     

A new austenitic stainless steel with a negligible amount of nickel: an in vitro study in view of its clinical application in osteoporotic bone

A biomaterial named P558 is a new austenitic stainless steel (SS) with a negligible amount of Ni (<0.20%). In previous in vitro and in vivo studies it was compared with conventional SS and Ti6Al4V and shown to be a promising material in orthopedics. Because osteoporosis is a type of pathology very often encountered in implanted patients and can be studied with in vitro models, the purpose of the present study was to evaluate P558 in vitro through comparison of normal (nOB) with osteopenic (oOB) bone-derived primary rat osteoblasts. Osteoblasts were cultured directly on P558 and polystyrene as controls for 72 h. Osteoblast proliferation, adhesion, and activity (ALP, OC, TGF-beta1, and IL-6) were evaluated at 24 and 72 h. Results demonstrated that the growth of nOB and oOB cultured on P558 was not affected negatively when compared to control. Cells on P558 did not show any alteration in terms of adhesion, proliferation, and metabolic marker production in nOB and oOB cultures, and a significant increase in ALP, OC, and TGF-beta1 production was observed. SEM images revealed no alteration in cell morphology. The current findings demonstrate that P558 promotes osteoblast proliferation, activation, and differentiation not only in normal bone, but also in osteopenic bone-derived osteoblasts.     

Soft tissue response to a new austenitic stainless steel with a negligible nickel content

This study evaluates the soft tissue response to a new austenitic stainless steel with a low nickel content (P558) in comparison with a conventional stainless steel (SSt)and a titanium alloy (Ti6Al4V). Previous findings showed its in vitro biocompatibility by culturing P558 with healthy and osteoporotic osteoblasts and its in vivo effectiveness as bone implant material. Regarding its use as a material in osteosynthesis,P558 biocompatibility when implanted in soft tissues, as subcutis and muscle, was assessed. Disks and rods of these metals were implanted in rat subcutis and in rabbit muscle, respectively. Four and twelve weeks post surgery implants with surrounding tissue were retrieved for histologic and histomorphometric analysis: fibrous capsule thickness and new vessel formation were measured. Around all implanted materials, light microscopy highlighted a reactive and fibrous capsule formation coupled with ongoing neoangiogenesis both in rats and in rabbits. Histomorphometric measurements revealed a stronger inflammatory response,in terms of capsule thickness,surrounding SSt implants (9.8% Ni content) both in rat subcutis and in rabbit muscle independently of shape and site of implantation. A progressive decrease in capsule thickness around P558 (<0.02% Ni content) and Ti6Al4V, respectively, was seen. Regarding new vessel density, the data showed a different response dependent on the site of implantation. However,in the light of the previous and present studies, P558 is a good material, instead of titanium alloys, in orthopedic research.     

抗菌钛合金Ti6Al4V-6Cu表面构建成骨细胞膜片的实验研究

目的:探讨大鼠颌骨成骨细胞在抗菌钛合金Ti6Al4V-6Cu表面构建细胞膜片的可行性。方法:体外培养大鼠颌骨成骨细胞,采用富含维生素C培养基在抗菌钛合金Ti6Al4V-6Cu表面构建细胞膜片（细胞膜片组）,并以单纯培养基作对照（对照组）,检测膜片形成过程中碱性磷酸酶（ALP）和成骨相关基因ALP、I型胶原（Col-1）、骨形成蛋白2（BMP-2）的表达情况。结果:采用富含维生素C的培养基连续培养可在抗菌钛合金Ti6Al4V-6Cu表面成功构建成骨细胞膜片,该细胞膜片由多层细胞构成,富含胞外基质。相对于对照组,细胞膜片组的膜片形成过程中成骨细胞ALP活性及成骨相关基因ALP、Col-1、BMP-2的表达均显著增高。结论:在抗菌钛合金Ti6Al4V-6Cu表面可成功构建成骨细胞膜片,有望与Ti6Al4V-6Cu联合应用于引导性骨再生术。     

Osteoblast differentiation onto different biometals with an endoprosthetic surface topography in vitro

In this in vitro study, we compared the cytocompatibility and osteoblast promoting potency on human bone marrow cell culture with three different alloys (surgical steel, CoCr, Ti6Al4V) and three different surface structures (polished, sandblasted, porous coated). These biometals were specifically chosen because of their current applications in clinical orthopedic practices. Human mononuclear bone marrow cells were cultivated onto the surface of the different biomaterials and stimulated by dexamethasone, L-ascorbic-acid-2-phoshpate and beta-glycerolphosphate over a 3-week period. Immunofluorescent stainings against several antigens (ALP, RANKL, osteopontin, collagen I), mRNA-expression of collagen (Col) I/II, BSP, osteopontin, osteocalcin, TRAP, light and scanning electron microscopy evaluation were used to evaluate cellular growth and osteoblast differentiation. For surface roughness and energy analysis of the specimen, roughness profile (Ra, Rz) and contact angle (CA) measurements were performed. We found differences between the different biometals and surface structures. Steel showed potential cytotoxic effects whereas CoCr and more Ti6Al4V showed an excellent cytocompatibility. There were no qualitative differences in mRNA expression between each of the tested biomaterials. In terms of antigen expression, a sandblasted Ti6Al4V surface showed enhanced osteoblastic differentiation. A porous-coated surface improved the osteoconductivity of CoCr when compared to a polished surface. In contrast to controls all cells cultivated with biometals induced a RANKL expression. Cells increased the implant roughness with the exception of sandblasted Ti6Al4V. Our data show that surface topography and physicochemical properties of biometals influence osteoblast differentiation in vitro.     

Osteoblast response and osseointegration of a Ti–6Al–4V alloy implant incorporating strontium

This study investigated the surface characteristics, in vitro and in vivo biocompatibility of Ti–6Al–4V alloy implants incorporating strontium ions (Sr), produced by hydrothermal treatment using a Sr-containing solution, for future biomedical applications. The surface characteristics were evaluated by scanning electron microscopy, thin-film X-ray diffractometry, X-ray photoelectron spectroscopy, optical profilometry, contact angle and surface energy measurement and inductively coupled plasma-mass spectroscopy (ICP-MS). Human osteoblast-like cell (MG63) attachment, proliferation, alkaline phosphatase (ALP) activity, and quantitative analysis of osteoblastic gene expression on Sr-containing Ti–6Al–4V surfaces were compared with untreated Ti–6Al–4V surfaces. Fifty-six screw implants (28 control and 28 experimental) were placed in the tibiae and femoral condyles of seven New Zealand White rabbits. The osteoconductivity of Sr-containing Ti–6Al–4V implants was evaluated by removal torque testing and histomorphometric analysis after 4 weeks implantation. Hydrothermal treatment produced a crystalline SrTiO₃ layer. ICP-MS analysis showed that Sr ions were released from treated surfaces into the solution. Significant increases in ALP activity (P = 0.000), mRNA expressions of key osteoblast genes (osterix, bone sialoprotein, and osteocalcin), removal torque values (P < 0.05) and bone–implant contact percentages (P < 0.05) in both cortical and cancellous bone were observed for Sr-containing Ti–6Al–4V surfaces. The results indicate that the Sr-containing oxide layer produced by hydrothermal treatment may be effective in improving the osseointegration of Ti–6Al–4V alloy implants by enhancing differentiation of osteoblastic cells, removal torque forces and bone apposition in both cortical and cancellous bone.     

In search of representative models of human bone-forming cells for cytocompatibility studies

Osteosarcoma-derived cells have been routinely used for studying osteoblastic functions, but it remains unclear to what extent they mimic the behavior of primary osteoblasts in the study of cells and materials interactions. This study reports comparatively on the responses of three human osteosarcoma cell lines, MG-63, Saos-2 and U-2 OS, and human primary osteoblasts cultured on Ti6Al4V surfaces or exposed to Ti particles. Phenotypic characterization of the cell lines revealed that Saos-2 cells and primary osteoblasts displayed similar expression patterns of Cbfa1, SP7 and osteocalcin. Unlike primary cells, the cell lines expressed markers of undifferentiated cells, had high proliferative rates and poor fibronectin matrix assembly. None of the three cell lines faithfully reproduced the adhesive behavior of primary osteoblasts when cultured on Ti6Al4V surfaces or exposed to Ti particles. Differences in cell growth between the cell lines and primary osteoblasts cultured on Ti6Al4V surfaces were also observed. Ti particles inhibited the growth of Saos-2 cells and primary osteoblasts to a similar extent, while no such effect was observed in U-2 OS and MG-63 cells. Saos-2 cells reproduced the alkaline phosphatase (ALP) activity profile of primary osteoblasts cultured on metallic surfaces or exposed to particles. Altogether, these results show that none of the osteoblast-like cells studied perfectly mimic the behavior of human osteoblast cells (hOB) on Ti6Al4V surfaces or exposed to Ti particles. Saos-2 cells reproduce some of the hOB responses such as the profile of enzymatic ALP activity when cultured on the surfaces or treated with particles as well as cell growth inhibition when exposed to Ti particles. Although in vitro cytocompatibility studies involve the evaluation of multiple parameters, Saos-2 cells may be used as representative of human osteoblasts when these standard tests are evaluated.     

Multi-level factorial analysis of Ca2+/Pi supplementation as bio-instructive media for in vitro biomimetic engineering of three-dimensional osteogenic hybrids

We report on the in vitro use of Ca(2+)/P(i) supplementation as a bio-instructive medium to drive human periosteum-derived cells (hPDCs) toward osteogenic differentiation on three-dimensional (3D) porous Ti6Al4V scaffolds. Through a multilevel factorial analysis, we have systematically investigated the biological effect and interactions of Ca(2+) or P(i) supplementation in three selected media preparations (i.e., basic growth medium, osteogenic medium [OM], and osteogenic medium without β-glycerophosphate [OM(-)]) and have identified specific conditions which induce proliferation and significant osteogenic differentiation of two-dimensional (2D) hPDC cultures. These findings were translated from 2D to 3D cultures conditions to instruct hPDCs to populate porous Ti6Al4V scaffolds and to differentiate into the osteoblast lineage with collagenous matrix production and subsequent matrix mineralization on the 3D structures. These osteogenic hybrids may potentially serve as a clinically relevant customizable bone reparative unit, providing a biomimetic template to more effectively mediate in vivo bone regeneration.     

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.     

Use of a novel carbon fibre composite material for the femoral stem component of a THR system: in vitro biological assessment

A novel, low elastic modulus femoral component for THR has been developed using a composite of polyetheretherketone and carbon fibre. The objectives of this study were to investigate human osteoblast-like cell and macrophage responses to this material in vitro. Cells were grown on composite discs and controls. Osteoblast attachment and proliferation was not significantly different to that on Ti6Al4V. The levels of alkaline phosphatase activity, Type I collagen production and osteocalcin production were not significantly different to that on Ti6Al4V by the end of the experimental period. Hydrogen peroxide production by macrophages was significantly less than that detected for cells cultured on copper, but was still greater than that detected for cells cultured on tissue culture plastic and Ti6Al4V. Beta-glucoronidase activity was not significantly different to that detected for cells cultured on tissue culture plastic. The in vitro biocompatibility assessment of this composite undertaken in this study showed initial osteoblast attachment at least comparable to that of the tissue culture plastic and Ti6Al4V controls, with proliferation similar to the controls at all time points up to 11 days. Alkaline phosphatase activity was similar to that of Ti6Al4V but reduced compared to tissue culture plastic controls. Whilst hydrogen peroxide production by macrophages was raised on composite surfaces compared to controls, beta-glucoronidase activity and osteoblastic production of Type I collagen and osteocalcin were similar to levels detected on Ti6Al4V.     

Biomimetic PMMA-based bone substitutes: a comparative in vitro evaluation of the effects of pulsed electromagnetic field exposure

Pulsed electromagnetic fields (PEMFs) are known to be effective in the stimulation of cultured osteoblasts and in vivo healing of delayed and nonunion fractures. In the present in vitro study the effects of PEMFs on osteoblastic cell cultures (MG63 human osteoblast-like cells) grown in the presence of poly-methylmethacrylate (PMMA) and of a biomimetic bone substitute made of a PMMA matrix added with alfa-tricalcium phosphate (PMMA+alpha-TCP) were evaluated, to assess the biological response at the cell-biomaterial interaction. Cultures were stimulated with PEMFs (75 Hz, 2.3 mT, 1.3-ms pulse duration) 12 h/day for 3 days and evaluations (MTT, ALP, OC, PICP, TGFbeta-1, IL-6) were performed at 3 and 6 days. PMMA had a negative effect on osteoblasts, whereas PMMA+alpha-TCP enhanced production of ALP, PICP, OC and TGFbeta-1, and reduced IL-6 levels. Cells responded positively to PEMF stimulation even when cultured with a poorly biocompatible material, such as PMMA. This effect was more evident in the presence of PMMA+alpha-TCP (further improvement in proliferation and synthetic activity) both at 3 and at 6 days. The properties of PMMA+alpha-TCP look promising, and the present results support the use of PEMFs to improve tissue response to biomaterials implanted as bone substitutes.     

Contact profilometry and correspondence analysis to correlate surface properties and cell adhesion in vitro of uncoated and coated Ti and Ti6Al4V disks

A fundamental goal in the field of implantology is the design of specific devices able to induce a controlled and rapid "osseointegration". This result has been achieved by means of surface modifications aimed at optimizing implant-to-bone contact; furthermore, bone cell adhesion on implant surface has been directly improved by the application of biomolecules that stimulate new tissue formation, thus controlling interactions between biological environment and implanted materials. Actually, methods for biochemical factor delivery at the interface between implant surface and biological tissues are under investigation; a reliable technique is represented by the inclusion of biologically active molecules into biocompatible and biodegradable materials used for coating implant surface. This paper focuses the application of three polymeric materials already acknowledged in the clinical practice, i.e. poly-L-lactic acid (PLLA), poly-DL-lactic acid (PDLA), and sodium alginate hydrogel. They have been used to coat Ti (Ti2) and Ti6Al4V (Ti5) disks; their characteristics have been determined and their performances compared, with specific regard to the ability in allowing osteoblast adhesion in vitro. Moreover, profilometry data analysis permitted to identify a specific roughness parameter (peak density) which mainly controls the amount of osteoblast adhesion.     

Concentration-dependent effects of titanium and aluminium ions released from thermally oxidized Ti6Al4V alloy on human osteoblasts

Thermal oxidation treatments of Ti6Al4V, at 500 and 700 degrees C, for 1 h result in the formation of an outer "ceramic" layer of rutile, which enhances osteoblast response. In the present study, we have measured in vitro Ti and Al ion release from Ti64 alloy in the as-received state and after thermal oxidation treatments at 500 or 700 degrees C, to culture medium under standard cell-culture conditions. Concentrations of both Ti and Al released from both thermal oxidation treatments were lower than from polished alloy. Al was released from the treated or untreated surfaces in substantially lower extent than Ti. Titanium and aluminium ions affected primary human osteoblast proliferation, metabolic activity, and differentiation in a dose-dependent manner. Treatments with individual Ti or Al metal ions in similar concentration ranges than released from the surfaces did not alter osteoblast response, which also remained unaffected after treatments with combinations of Ti plus Al applied in the proportional relations than detected in ion-release experiments. We then selected higher concentrations of Ti that impaired osteoblast proliferation and differentiation, while the proportional lower concentrations of Al did not alter osteoblast behavior. In spite of its inert character, it was found that Al significantly enhanced the deleterious effect of Ti on osteoblast differentiation. Therefore, thermal oxidation treatments of Ti6Al4V alloy may improve the biocompatibility of the alloy by reducing both Ti and Al release, and thus attenuating ion-mediated interference with osteoblast differentiation.     

Calcium phosphate-chitosan composite scaffolds for bone tissue engineering

Macroporous calcium phosphate-chitosan composite scaffolds were fabricated and evaluated for use in bone tissue engineering. Human osteoblast-like MG63 cells were cultured on the composite scaffolds, and their response to the materials was studied. Cell morphology, total protein content, and expression of classic markers for osteoblast differentiation were characterized. MG63 cells on the hydroxyapatite scaffolds nesting chitosan sponges (HC1) showed significantly higher alkaline phosphatase (ALP) level and osteocalcin (OC) production during the 11-day culture period, compared with the control culture on tissue culture plates. Cells on the chitosan scaffolds incorporated with hydroxyapatite powders (HC2) exhibited lower ALP activity during the 11-day culture period and OC secretion during the first 7 days, in comparison with that on HC1. The addition of calcium phosphate glass as in HC3 scaffolds increased the ALP and OC levels of MG63 cells. Our study indicated that the hydroxyapatite-matrix composite scaffolds might enhance the phenotype expression of MG63 cells, in comparison with chitosan-matrix scaffolds. Soluble calcium phosphate glasses should be added to the scaffolds to prevent chitosan from fast degradation that may affect the differentiation of osteoblast cells.     

Biological performance of uncoated and octacalcium phosphate-coated Ti6Al4V

The in vivo behavior of a porous Ti6Al4V material that was produced by a positive replica technique, with and without an octacalcium phosphate (OCP) coating, has been studied both in the back muscle and femur of goats. Macro- and microporous biphasic calcium phosphate (BCP) ceramic, known to be both osteoconductive and able to induce ectopic bone formation, was used for comparison purpose. The three groups of materials (Ti6Al4V, OCP Ti6Al4V and BCP) were implanted transcortically and intramuscularly for 6 and 12 weeks in 10 adult Dutch milk goats in order to study their osteointegration and osteoinductive potential. In femoral defects, both OCP Ti6Al4V and BCP were performing better than the uncoated Ti6Al4V, at both time points. BCP showed a higher bone amount than OCP Ti6Al4V after 6 weeks of implantation, while after 12 weeks, this difference was no longer significant. Ectopic bone formation was found in both OCP Ti6Al4V and BCP implants after 6 and 12 weeks. The quantity of ectopically formed bone was limited as was the amount of animals in which the bone was observed. Ectopic bone formation was not found in uncoated titanium alloy implants, suggesting that the presence of calcium phosphate (CaP) is important for bone induction. This study showed that CaPs in the form of coating on metal implants or in the form of bulk ceramic have a significantly positive effect on the bone healing process.     

Thermal oxidation enhances early interactions between human osteoblasts and alumina blasted Ti6Al4V alloy

Oxidation of Ti6Al4V at 500 degrees C for 1 h in air results in the formation of an outer ceramic layer that improves osteoblast behavior and decreases Ti and Al ion release. In this work, alumina blasted Ti6Al4V alloy has been thermally treated and its in vitro biocompatibility has been assessed. Roughness of the blasted alloy was not found significantly altered after heat treatment while chemical surface analysis indicated an increase in stable TiO(2) and Al(2)O(3) oxides. Cell attachment, spreading, cytoskeleton organization as well as cell proliferation, viability, and procollagen I peptide secretion of human primary osteoblasts, impaired on alumina blasted Ti6Al4V, were found to be greatly enhanced on the thermally oxidized blasted alloy. Other informative markers of the osteoblastic phenotype such as alkaline phosphatase, osteocalcin, osteoprotegerin, and mineralized nodule formation were evaluated and indicated that osteoblasts responded at the same extent on untreated and thermally treated blasted alloys. Taken together, our in vitro results indicate that thermal oxidation of alumina blasted Ti6Al4V may favor successful osseointegration by promoting early interactions of osteoblastic cells and the modified surface alloy.     

Enhanced osteoblast response to an equal channel angular pressing-processed pure titanium substrate with microrough surface topography

This study investigated the surface characteristics and in vitro biocompatibility of ultrafine-grain pure titanium substrates produced by equal channel angular pressing (ECAP) using MC3T3-E1 pre-osteoblast cells, compared with those of conventional coarse-grain pure titanium (CP) and Ti–6Al–4V (Ti64) substrates. All Ti surfaces were grit-blasted with hydroxyapatite particles to produce microrough surfaces. The surface characteristics were evaluated by electron back-scattered diffractometry, scanning electron microscopy, contact angle and surface energy measurements, and optical profilometry. The morphology of spread cells, cell attachment, viability, alkaline phosphatase (ALP) activity, quantitative analysis of osteoblastic gene expression and mineralization nodule formation on different surfaces were evaluated. ECAP-processed substrates showed a significantly lower water contact angle and higher surface energy compared with coarse-grain CP and Ti64 substrates (p < 0.05). They also showed enhanced cell spreading, attachment, viability and ALP activity compared with the CP and Ti64 surfaces (p < 0.05). Real-time polymerase chain reaction analysis showed notably higher ALP, osteopontin and osteocalcin mRNA levels in cells grown on the ECAP surfaces than on the CP and Ti64 surfaces, and the ECAP surfaces showed significantly greater mineralization nodule formation compared with the CP and Ti64 substrates (p < 0.05). These results demonstrate the superior osteoblast cell compatibility of microroughened Ti surface made of ECAP-processed ultrafine-grain pure Ti substrates over coarse-grain pure Ti and Ti64 substrates.     

Isolation and characterization of osteoblast cultures from normal and osteopenic sheep for biomaterials evaluation

Being very useful in the analysis of bone cell differentiation and activity, osteoblast cultures are also used in the in vitro biocompatibility study of new materials. The aim of this work was to evaluate sheep osteoblast cultures derived from normal and ovariectomized animals, and then to assess the in vitro biomaterial behavior on these cultures, taking into account the quality of bone where orthopedic devices are clinically used. For this purpose, we characterized sheep osteoblast cultures, isolated from iliac crest bone of normal (NB osteoblast culture) and osteopenic after ovariectomy (OB osteoblast culture) sheep. Moreover, we studied cell behavior when cultured on different biomaterials (titanium and two biological glasses, RKKP and AP40). Cell characterization at baseline demonstrated that both cultures (NB and OB) showed normal osteoblastic behavior. On the contrary, osteoblasts derived from osteopenic bone and cultivated on AP40 for 6 days revealed a different behavior in terms of both cell morphology and metabolic activity. Statistical analysis (one-way analysis of variance and Scheffé's post hoc multiple-comparison tests) revealed significant differences in Ca level (p<0.0005), MTT test (p<0.0005) and OC production (p<0.05). These in vitro tests demonstrated that sheep osteoblast cultures can be useful when determining biocompatibility and osteointegration of orthopedic materials, and also when evaluating for the presence of osteoporosis.     

Human osteopenic bone-derived osteoblasts: essential amino acids treatment effects

The development of in vitro cell culture methods has made it possible to study bone cell metabolism and growth and obtain a deeper insight into the pathophysiology of common orthopedic diseases such as osteoporosis. After analyzing the effect of two essential amino acids, L-arginine (Arg) and L-lysine (Lys), in previous in vitro and in vivo studies, the present authors investigated the administration of Arg and Lys in osteoblasts derived from human osteopenic bone. After isolation, osteoblasts were cultured in DMEM supplemented with either Arg (0.625 mg/ml/day, Arg Group) or Lys (0.587 mg/ml/day, Lys Group), or both of them (Arg-Lys Group), whereas the Control Group was sham-treated. After 7 days the following parameters were tested in all groups: MTT proliferation test, Alkaline Phosphatase (ALP), Nitric Oxide (NO), Calcium (Ca), Phosphorus (P), Osteocalcin (OC), C-Terminal Procollagen type I (PICP), Interleukin-6 (IL-6), Transforming Growth Factor-beta 1 (TGF-beta 1), Platelet Derived Growth Factor (PDGF) and Insulin-Like Growth Factor-I (IGF-I). Results were compared with those obtained from human healthy bone to verify the effect of the amino acids on osteoblasts derived from pathological tissue. In addition, a comparison was also made with the results obtained from rat osteopenic bone to assess reliability of the in vitro model. The current results support previous findings and indicate that Arg and Lys stimulation has a positive effect on osteoblast proliferation, activation and differentiation. Therefore, administration of these amino acids may be useful in clinical treatment and prevention of osteoporosis.