Tissue engineering (TE) has emerged as a promising new therapy for the treatment of damaged tissues and organs. Adult stem cells are considered as an attractive candidate cell type for cell-based TE. Mesenchymal stem cells (MSC) have been isolated from a variety of tissues and tested for differentiation into different cell lineages. While clinical trials still await the use of human MSC, horse tendon injuries are already being treated with autologous bone marrow-derived MSC. Given that the bone marrow is not an optimal source for MSC due to the painful and risk-containing sampling procedure, isolation of stem cells from peripheral blood would bring an attractive alternative. Adherent fibroblast-like cells have been previously isolated from equine peripheral blood. However, their responses to the differentiation conditions, established for human bone marrow MSC, were insufficient to fully confirm their multilineage potential. In this study, differentiation conditions were optimized to better evaluate the multilineage capacities of equine peripheral blood-derived fibroblast-like cells (ePB-FLC) into adipogenic, osteogenic, and chondrogenic pathways. Adipogenic differentiation using rabbit serum resulted in a high number of large-size lipid droplets three days upon induction. Cells' expression of alkaline phosphatase and calcium deposition upon osteogenic induction confirmed their osteogenic differentiation capacities. Moreover, an increase of dexamethasone concentration resulted in faster osteogenic differentiation and matrix mineralization. Finally, induction of chondrogenesis in pellet cultures resulted in an increase in cartilage-specific gene expression, namely collagen II and aggrecan, followed by protein deposition after a longer induction period. This study therefore demonstrates that ePB-FLC have the potential to differentiate into adipogenic, osteogenic, and chondrogenic mesenchymal lineages. The presence of cells with confirmed multilineage capacities in peripheral blood has important clinical implications for cell-based TE therapies in horses.

Chondromyxoid fibroma is a rare benign cartilaginous bone tumour characterized by morphological features that resemble different steps of chondrogenesis in terms of both cellular morphology, ranging from spindled to rounded cells, and the extracellular matrix formed, which ranges from fibrous to cartilaginous. The presence in chondromyxoid fibroma of signalling molecules that regulate the spatial expression of proteins involved in normal cartilage proliferation and differentiation was investigated in samples from 20 patients and compared with articular chondrocytes from 11 normal donors cultivated in 3D pellet culture. Sections were stained with safranin-O and H&E, and immunohistochemistry was performed for p16, cyclin D1, FGFR3, BCL2, p21, PTHLH, PTHR1 and N-cadherin. Expression patterns were analysed using hierarchical clustering. In chondromyxoid fibroma, specific morphological features correlated with a distinct pattern of expression. Comparison with normal chondrocytes in pellet culture showed a striking morphological resemblance, but with an unmistakably different pattern of expression. N-cadherin, PTHLH, and PTHR1 were expressed to a significantly higher level (p < 0.01) in articular chondrocyte pellets but, conversely, there was significantly lower expression of cyclin D1, p16 and BCL2 (p < 0.05) in these cells. Morphological similarities reflect common steps in cartilage differentiation, albeit driven by different molecular mechanisms. The proteins we have found to be differentially expressed seem crucial for neoplastic chondrogenesis. Copyright © 2005 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Objective: To compare the in vitro osteogenic differentiation and in vivo ectopic bone forming capacity of human bone marrow stromal cells (BMSCs) and jaw periosteal cells (JPCs), and to identify molecular predictors of their osteogenic capacity. Summary Background Data: JPC could be an appealing alternative to BMSC for the engineering of cell-based osteoinductive grafts because of the relatively easy access to tissue with minimal morbidity. However, the extent of osteogenic capacity of JPC has not yet been established or compared with that of BMSC. Methods: BMSCs and JPCs from the same donors (N ϭ 9), expanded for 2 passages, were cultured for 3 weeks in osteogenic medium either in monolayers (Model I) or within 3-dimensional porous ceramic scaffolds, following embedding in fibrin gel (Model II). Cell-fibrin-ceramic constructs were also implanted ectopically in nude mice for 8 weeks (Model III). Cell differentiation in vitro was assessed biochemically and by real-time RT-PCR. Bone formation in vivo was quantified by computerized histomorphometry. Results: JPCs had lower alkaline phosphatase activity, deposited smaller amounts of calcium (Model I), and expressed lower mRNA levels of bone sialoprotein, osteopontin, and osterix (Models I and II) than BMSCs. JPCs produced ectopic bone tissue at lower frequency and amounts (Model III) than BMSCs. Bone sialoprotein, osteopontin, and osterix mRNA levels by BMSCs or JPCs in Model II were markedly higher than in Model I and significantly more expressed by cells that generated bone tissue in Model III. Conclusions: Our data indicate that JPCs, although displaying features of osteogenic cells, would not be as reliable as BMSCs for cell-based bone tissue engineering, and suggest that expression of osteoblast-related markers in vitro could be used to predict whether cells would be osteoinductive in vivo. (Ann Surg 2005;242: 859 -868) From the *

Fibroblast-like cells isolated from peripheral blood of human, canine, guinea pig, and rat have been demonstrated to possess the capacity to differentiate into several mesenchymal lineages. The aim of this work was to investigate the possibility of isolating pluripotent precursor cells from equine peripheral blood and compare them with equine bone marrow-derived mesenchymal stem cells. Human mesenchymal stem cells (MSCs) were used as a control for cell multipotency assessment. Venous blood (n = 33) and bone marrow (n = 5) were obtained from adult horses. Mononuclear cells were obtained by Ficoll gradient centrifugation and cultured in monolayer, and adherent fibroblast-like cells were tested for their differentiation potential. Chondrogenic differentiation was performed in serum-free medium in pellet cultures as a three-dimensional model, whereas osteogenic and adipogenic differentiation were induced in monolayer culture. Evidence for differentiation was made via biochemical, histological, and reverse transcription-polymerase chain reaction evaluations. Fibroblast-like cells were observed on day 10 in 12 out of 33 samples and were allowed to proliferate until confluence. Equine peripheral blood-derived cells had osteogenic and adipogenic differentiation capacities comparable to cells derived from bone marrow. Both cell types showed a limited capacity to produce lipid droplets compared to human MSCs. This result may be due to the assay conditions, which are established for human MSCs from bone marrow and may not be optimal for equine progenitor cells. Bone marrow-derived equine and human MSCs could be induced to develop cartilage, whereas equine peripheral blood progenitors did not show any capacity to produce cartilage at the histological level. In conclusion, equine peripheral blood-derived fibroblast-like cells can differentiate into distinct mesenchymal lineages but have less multipotency than bone marrow-derived MSCs under the conditions used in this study.

OBJECTIVE: The present study not only investigates cartilage structure and biochemistry, but also tracks the progressive changes in bone density, subchondral trabecular bone architecture, bone turnover, mechanical and material properties of bone, and bone mineralization of both traumatic and accelerated spontaneous OA in a single animal model from time 0 to 10, 20, 30, 90, and 180 days after surgery. METHODS: Sixty guinea pigs were used in this study. Fifty 2-months old male Dunkin-Hartley guinea pigs underwent partial medial meniscectomy (pMNX) in the right knee. Ten animals were euthanized at 0, 10 and 20days, 1, 3, and 6 months time points postsurgery OA cartilage morphological changes were validate/evaluated by Mankin grading. Cartilage biochemistry, bone mineral density (BMD), subchondral bone plate thickness, bone histomorphometry and osteophyte formation were studied.RESULTS: Animals underwent partial medial meniscectomy displayed significantly higher degree of cartilage degeneration, increased subchondral bone turnover, early decrease followed by an increase in bone mineral density and bone volume and subchondral bone plate area. RESULT: In this guinea pig model, we have observed spontaneous OA-like changes in the un-operated knee, and accelerated progressive changes in the operated knee. Cartilage structure changes include: surface destruction, chondrocyte hyper/hypocellularity, tidemark duplication, and loss of cartilage. Changes in cartilage biochemistry include: loss of proteoglycan, collagen, and DNA. Subchondral bone changes include: calcified cartilage thickening, changes in subchondral bone plate thickness and mineralization, changes in trabecular bone remodeling, architecture, and material properties. CONCLUSION: The accelerated nature of this surgery-enhanced spontaneous OA model has been confirmed by the rapid occurrence of bone and cartilage changes in the operated knees while similar changes were observed in the contralateral joint only at later times. Therefore, this is a suitable model to study the epidemiology, disease progression and treatment of OA.

Pain in the joint is often due to cartilage degeneration and represents a serious medical problem affecting people of all ages. Although many, mostly surgical techniques, are currently employed to treat cartilage lesions, none has given satisfactory results in the long term. Recent advances in biology and material science have brought tissue engineering to the forefront of new cartilage repair techniques. The combination of autologous cells, specifically designed scaffolds, bioreactors, mechanical stimulations and growth factors together with the knowledge that underlies the principles of cell biology offers promising avenues for cartilage tissue regeneration. The present review explores basic biology mechanisms for cartilage reconstruction and summarizes the advances in the tissue engineering approaches. Furthermore, the limits of the new methods and their potential application in the osteoarthritic conditions are discussed.

Biological restoration of osteochondral defects requires suitable subchondral support material that also allows the induction of hyaline cartilage tissue. Biphasic implants consisting of prefabricated neocartilage and an underlying biodegradable osteoconductive base may meet these requirements. Here we explore various candidate biodegradable support materials onto which neo-cartilage was produced in vitro. Porcine chondrocytes were seeded in a closed and static bioreactor with a base of biomaterial consisting of either poly-l-lactide [P(l)LA], poly-d,l-lactide [P(d,l)LA] or Collagen-hydroxyapatite [Col-HA] and were cultured for 15 weeks. Viable neo-cartilage was produced on each biomaterial with differing amounts of cellular colonisation. P(d,l)LA breakdown was more rapid and uneven among the three biomaterials, leading to constructs of irregular shape. Little or no breakdown or chondrocyte colonisation was evident in P(l)LA. Col-HA constructs were superior in terms of viability, implant morphology and integration between neo-cartilage and biomaterial. These results indicate that our reported system has potential for producing biphasic implants that may be adequate for the repair of osteochondral defects.

Cartilage repair strategies aim to resurface a lesion with osteochondral tissue resembling native cartilage, but a variety of repair tissues are usually observed. Histology is an important structural outcome that could serve as an interim measure of efficacy in randomized controlled clinical studies. The purpose of this article is to propose guidelines for standardized histoprocessing and unbiased evaluation of animal tissues and human biopsies. Methods were compiled from a literature review, and illustrative data were added. In animal models, treatments are usually administered to acute defects created in healthy tissues, and the entire joint can be analyzed at multiple postoperative time points. In human clinical therapy, treatments are applied to developed lesions, and biopsies are obtained, usually from a subset of patients, at a specific time point. In striving to standardize evaluation of structural endpoints in cartilage repair studies, 5 variables should be controlled: 1) lo...

Investigational devices for articular cartilage repair or replacement are considered to be significant risk devices by regulatory bodies. Therefore animal models are needed to provide proof of efficacy and safety prior to clinical testing. The financial commitment and regulatory steps needed to bring a new technology to clinical use can be major obstacles, so the implementation of highly predictive animal models is a pressing issue. Until recently, a reductionist approach using acute chondral defects in immature laboratory species, particularly the rabbit, was considered adequate; however, if successful and timely translation from animal models to regulatory approval and clinical use is the goal, a step-wise development using laboratory animals for screening and early development work followed by larger species such as the goat, sheep and horse for late development and pivotal studies is recommended. Such animals must have fully organized and mature cartilage. Both acute and chronic...

A n issue that confronts the top management of ecery multinational company is that ofdetermining where in the organization basic strategy decisions should be made. Thefirm may concentrate responsibility for a broad range of strategic tasks at headquarters, or cede strategic responsibility to foreign afjiliates. This paper argues that deriving an appropriate apportionment of strategic responsibility is largely a matter of recognizing the unique strategic imperatives imposed on the multinational firm by the conflicting requirements of integration within a global contexl, and responsiveness to local environments. For the firm whose businesses can benefit f r o m worldwide coordination and standardization of manufacturing andproduct decelopment, there is strong pressure to consolidate strategic responsibility at headquarters. For the firm whose businesses requireattention to the idiosyncracies ofnational markets, the strategic autonomy of subsidiaries may be requisite for success. A variety of factors renders diJicult a clear-cut, one-time identiJcation of the strategic imperatice facing a business. For example, even within a single business, certain functions respond to the logic of integration, whereas others require a posture of responsiveness. At times both imperatices exist at full strength within a single functional area. Often, in a multi-business, multinationalfirm, different businesses aresubject to different pressures f o r integration and responsiveness. It is shown that managing strategic responsibility in situations of such ambiguity forces the firm to look far beyond traditional structural mechanisms in order to enact an appropriate division of strategic responsibility between headquarters and affiliates.

The fundamental structural transitions in a wide variety of industries brought about by major catalysts such as deregulation, global competition, technological discontinuities, and changing customer expectations are imposing new strains on managers around the world. Old recipes do not work anymore. Managers, concerned with restoring competitiveness of their firms, are abandoning traditional approaches to strategy; they are searching for new approaches that give guidance in a turbulent environment. Many academics, confronted with the same reality, are reexamining the relevance of the concepts and tools of the strategy field. In the absence of a consistent and useful strategy paradigm that they can use, managers appear to have embraced attention to 'implementation' as their saviour, more or less abandoning strategy as either unimportant or uninteresting. Academics continue to search for new approaches. This special issue of the Strategic Management Journal presents creative and new thinking dealing with substantive issues and methodologies that can lead to the evolution of a new paradigm(s). As we entered the 1990s, strategy as a field of study had fallen on hard times. Humbled by new global competitors, managers were consumed with TQM, reengineering, downsizing, teamwork and employee empowerment. Managerial preoccupation was with 'catching up' with the best of breed among their competitors. Issues of strategy seemed either remote, unimportant or uninteresting to many. The key words were 'implementation' and 'execution'. Strategy, some managers seemed to assume, was easy; implementation was the hard part. As a consequence, strategy staffs were dramatically reduced or eliminated. Consultants readjusted their wares to cope with the new client demands. Even wellknown consulting firms, such as McKinsey and Boston Consulting Group (BCG), who built their Key words: Strategy paradigms, innovative strategy research, paradigm evolution I See, for example, the focus of BCG on 'Time Based Competition'. Two senior consultants of BCG wrote the very popular book on the subject, George Stalk, Jr. and Thomas M. Hout, (1990). Competing Against Time: How Time Based Competition is Reshaping Global Markers, Free Press, New York.