Coefficient of Friction Patterns Can Identify Damage in Native and Engineered Cartilage Subjected to Frictional-Shear Stress.

The mechanical loading environment encountered by articular cartilage in situ makes frictional-shear testing an invaluable technique for assessing engineered cartilage. Despite the important information that is gained from this testing, it remains under-utilized, especially for determining damage behavior. Currently, extensive visual inspection is required to assess damage; this is cumbersome and subjective. Tools to simplify, automate, and remove subjectivity from the analysis may increase the accessibility and usefulness of frictional-shear testing as an evaluation method. The objective of this study was to determine if the friction signal could be used to detect damage that occurred during the testing. This study proceeded in two phases: first, a simplified model of biphasic lubrication that does not require knowledge of interstitial fluid pressure was developed. In the second phase, frictional-shear tests were performed on 74 cartilage samples, and the simplified model was used to extract characteristic features from the friction signals. Using support vector machine classifiers, the extracted features were able to detect damage with a median accuracy of approximately 90%. The accuracy remained high even in samples with minimal damage. In conclusion, the friction signal acquired during frictional-shear testing can be used to detect resultant damage to a high level of accuracy.

Water-in-trichloroethylene emulsions stabilized by uniform carbon microspheres.

Uniform hard carbon spheres (HCS), synthesized by the hydrothermal decomposition of sucrose followed by pyrolysis, are effective at stabilizing water-in-trichloroethylene (TCE) emulsions. The irreversible adsorption of carbon particles at the TCE-water interface resulting in the formation of a monolayer around the water droplet in the emulsion phase is identified as the key reason for emulsion stability. Cryogenic scanning electron microscopy was used to image the assembly of carbon particles clearly at the TCE-water interface and the formation of bilayers in regions of droplet-droplet contact. The results of this study have potential implications to the subsurface injection of carbon submicrometer particles containing zero-valent iron nanoparticles to treat pools of chlorinated hydrocarbons that are sequestered in fractured bedrock.

Optimization of the Expansion and Differentiation of Rabbit Chondrocytes In Vitro.

OBJECTIVE: To develop a tissue culture expansion method for rabbit chondrocytes that promotes robust expansion while preserving chondrogenic potential. DESIGN: Rabbit chondrocytes isolated from articular or auricular chondrocytes were assessed for chondrogenic differentiation potential versus population doubling using different expansion and differentiation conditions. Expansion conditions included serum alone, serum plus basic fibroblast growth factor 2 (FGF-2), and serum plus insulin-like growth factor 1 (IGF-1) and FGF-2. Differentiation conditions consisted of defined medium with and without bone morphogenetic protein 2 (BMP-2). RESULTS: Nonsupplemented chondrocytes showed limited expandability, whereas supplementation with FGF-2 allowed articular chondrocytes to be expanded past 10 population doublings (PDs) and allowed auricular chondrocytes to expand past 15 population doublings. Differentiation, as measured by glycosaminoglycan production in aggregate cultures, was minimal in articular chondrocytes without BMP-2 supplementation and diminished to less than 50% maximal in auricular chondrocytes by PD 20. However, when FGF-2 was used during expansion and BMP-2 used during differentiation, both articular and auricular chondrocytes retained greater than 50% maximal differentiation for more than 25 PDs. The addition of IGF-1 to FGF-2 during expansion decreased chondrogenicity of auricular chondrocytes exposed to BMP-2, whereas for articular chondrocytes, chondrogenic expression increased. CONCLUSION: These results demonstrate that FGF-2, for expansion, and BMP-2, for differentiation, dramatically increase the functional expansion of auricular and articular chondrocytes and provide a methodology to expand sufficient numbers of chondrocytes for tissue engineering applications.

Carbon microspheres as ball bearings in aqueous-based lubrication.

We present an exploratory study on a suspension of uniform carbon microspheres as a new class of aqueous-based lubricants. The surfactant-functionalized carbon microspheres (∼0.1 wt %) employ a rolling mechanism similar to ball bearings to provide low friction coefficients (µ ≈ 0.03) and minimize surface wear in shear experiments between various surfaces, even at high loads and high contact pressures. The size range, high monodispersity, and large yield stress of the C(µsphere), as well as the minimal environmental impact, are all desirable characteristics for the use of a C(µsphere)-SDS suspension as an alternative to oil-based lubricants in compatible devices and machinery.

Phenotypic plasticity of human articular chondrocytes.

BACKGROUND: Progenitor cells in mesenchymal tissues are important in the maintenance of tissue homeostasis and regeneration capacity. Articular cartilage is a tissue with a very low capacity for repair. One explanation could be the lack of chondrogenic progenitor cells within the adult tissue. As a test of chondrogenic differentiation potential, we examined the ability of isolated chondrocytes to take on several phenotypic identities within the mesenchymal lineage by applying culture techniques and markers used in the study of the phenotypic plasticity of marrow-derived mesenchymal stem cells (MSCs). METHODS: Culture-expanded human articular chondrocytes were analyzed for chondrogenic, adipogenic, and osteogenic capacity in defined in vitro culture systems. The osteochondrogenic potential of cells loaded into porous calcium-phosphate ceramic cubes implanted into mice was also determined. RESULTS: The different assays demonstrated that culture-expanded chondrocytes have the potential to form cartilage in pellet mass cultures, to form adipose cells in dense monolayer cultures, and to form a calcium-rich matrix in an osteogenic assay. In the in vitro assays, a variability of phenotypic plasticity was demonstrated among the donors. In contrast with MSCs, chondrocytes formed cartilage only (and not bone) in the in vivo osteochondrogenic assay. CONCLUSIONS: These results suggest that, within articular cartilage, there are chondrogenic cells that exhibit a level of phenotypic plasticity that is comparable with that of MSCs. However, there was a difference in the expression of bone in the in vivo assay.

Tissue-engineered fabrication of an osteochondral composite graft using rat bone marrow-derived mesenchymal stem cells.

This study tested the tissue engineering hypothesis that construction of an osteochondral composite graft could be accomplished using multipotent progenitor cells and phenotype-specific biomaterials. Rat bone marrow-derived mesenchymal stem cells (MSCs) were culture-expanded and separately stimulated with transforming growth factor-beta1 (TGF-beta1) for chondrogenic differentiation or with an osteogenic supplement (OS). MSCs exposed to TGF-beta1 were loaded into a sponge composed of a hyaluronan derivative (HYAF-11) for the construction of the cartilage component of the composite graft, and MSCs exposed to OS were loaded into a porous calcium phosphate ceramic component for bone formation. Cell-loaded HYAFF-11 sponge and ceramic were joined together with fibrin sealant, Tisseel, to form a composite osteochondral graft, which was then implanted into a subcutaneous pocket in syngeneic rats. Specimens were harvested at 3 and 6 weeks after implantation, examined with histology for morphologic features, and stained immunohistochemically for type I, II, and X collagen. The two-component composite graft remained as an integrated unit after in vivo implantation and histologic processing. Fibrocartilage was observed in the sponge, and bone was detected in the ceramic component. Observations with polarized light indicated continuity of collagen fibers between the ceramic and HYAFF-11 components in the 6-week specimens. Type I collagen was identified in the neo-tissue in both sponge and ceramic, and type II collagen in the fibrocartilage, especially the pericellular matrix of cells in the sponge. These data suggest that the construction of a tissue-engineered composite osteochondral graft is possible with MSCs and different biomaterials and bioactive factors that support either chondrogenic or osteogenic differentiation.

The dynamic in vivo distribution of bone marrow-derived mesenchymal stem cells after infusion.

Bone marrow-derived mesenchymal stem cells (MSCs) have the potential to differentiate along different mesenchymal lineages including those forming bone, cartilage, tendon, fat, muscle and marrow stroma that supports hematopoiesis. This differentiation potential makes MSCs candidates for cell-based therapeutic strategies for mesenchymal tissue injuries and for hematopoietic disorders by both local and systemic application. In the present study, rat marrow-derived MSCs were ex vivo culture-expanded, labeled with (111)In-oxine, and infused into syngeneic rats via intra-artery (i.a.), intravenous (i.v.) and intraperitoneal cavity (i.p.) infusions. In addition, for i.a. and i.v. infusions, a vasodilator, sodium nitroprusside, was administered prior to the cell infusion and examined for its effect on MSC circulation. The dynamic distribution of infused MSCs was monitored by real-time imaging using a gamma camera immediately after infusion and at 48 h postinfusion. After 48 h, radioactivity in excised organs, including liver, lungs, kidneys, spleen and long bones, was measured in a gamma well counter and expressed as a percentage of injected doses. After both i.a. and i.v. infusion, radioactivity associated with MSCs was detected primarily in the lungs and then secondarily in the liver and other organs. When sodium nitroprusside was used, more labeled MSCs cleared the lungs resulting in a larger proportion detected in the liver. Most importantly, the homing of labeled MSCs to the marrow of long bones was significantly increased by the pretreatment with vasodilator. These results indicate multiple homing sites for injected MSCs and that the distribution of MSCs can be influenced by administration of vasodilator.

Monoclonal antibodies to mineralized matrix molecules of the avian eggshell.

The extracellular matrix of the mineralizing eggshell contains molecules hypothesized to be regulators of biomineralization. To study eggshell matrix molecules, a bank of monoclonal antibodies was generated that bound demineralized eggshell matrix or localized to oviduct epithelium. Immunofluorescence staining revealed several staining patterns for antibodies that recognized secretory cells: staining for a majority of columnar lining cells, staining for a minor sub-set of columnar lining cells, intensified staining within epithelial crypts, and staining of the entire tubular gland. Western blotting with the antibody Epi2 on eggshell matrix showed binding to molecules with the apparent molecular weight of eggshell matrix dermatan sulfate proteoglycan (eggshell DSPG). Immunoblots of cyanogen bromide-cleaved eggshell DSPG revealed broad band of reactivity that shifted to 25 kDa after chondroitinase digestion; indicating that the Epi2 binding site is located on a fragment which contains dermatan sulfate side chains. Immunogold labeling showed that Epi2 binds to secretory vesicles within the non-ciliated cells of the columnar epithelium, while the antibodies Tg1 and Tg2 bind to secretory vesicles of tubular gland cells. Immunogold labeling of demineralized shell matrix showed binding of Epi2, Tg1, and Tg2 to the matrix of the palisade layer, and showed little reactivity to other regions of the shell matrix. Quantification of the immunogold particles within the eggshell matrix revealed that antibodies Epi2 and Tg1 bind all calcified regions equally while antibody Tg2 has a greater affinity for the baseplate region of the calcium reserve assembly.

Hyaluronan-based polymers in the treatment of osteochondral defects.

Articular cartilage in adults has limited ability for self-repair. Some methods devised to augment the natural healing response stimulate some regeneration, but the repair is often incomplete and lacks durability. Hyaluronan-based polymers were tested for their ability to enhance the natural healing response. It is hypothesized that hyaluronan-based polymers recreate an embryonic-like milieu where host progenitor cells can regenerate the damaged articular surface and underlying bone. Osteochondral defects were made on the femoral condyles of 4-month-old rabbits and were left empty or filled with hyaluronan-based polymers. The polymers tested were ACP sponge, made of crosslinked hyaluronan, and HYAFF-11 sponge, made of benzylated hyaluronan. The rabbits were killed 4 and 12 weeks after surgery, and the condyles were processed for histology. All 12-week defects were scored with a 29-point scale, and the scores were compared with a Kruskall-Wallis analysis of variance on ranks. Untreated defects filled with bone tissue up to or beyond the tidemark, and the noncalcified surface layer varied from fibrous to hyaline-like tissue. Four weeks after surgery, defects treated with ACP exhibited bone filling to the level of the tidemark and the surface layer was composed of hyaline-like cartilage well integrated with the adjacent cartilage. At 12 weeks, the specimens had bone beyond the tidemark that was covered with a thin layer of hyaline cartilage. Four weeks after surgery, defects treated with HYAFF-11 contained a rim of chondrogenic cells at the interface of the implant and the host tissue. In general, the 12-week defects exhibited good bone fill and the surface was mainly hyaline cartilage. Treated defects received significantly higher scores than untreated defects (p < 0.05), and ACP-treated defects scored significantly higher than HYAFF-11-treated defects (p < 0.05). The introduction of these hyaluronan-based polymers into defects provides an appropriate scaffolding and favorable microenvironment for the reparative process. Further work is required to fully assess the long-term outcome of defects treated with these polymers.

A quadripotential mesenchymal progenitor cell isolated from the marrow of an adult mouse.

Adult marrow contains mesenchymal progenitor cells (MPCs) that have multiple differentiation potentials. A conditionally immortalized MPC clone, BMC9, has been identified that exhibits four mesenchymal cell phenotypes: chondrocyte, adipocyte, stromal (support osteoclast formation), and osteoblast. The BMC9 clone, control brain fibroblasts and another marrow-derived clone, BMC10, were isolated from a transgenic mouse (H-2Kb-tsA58) containing a gene for conditional immortality. To test for chondrogenic potential, cells were cultured in defined medium containing 10 ng/ml transforming growth factor beta and 10-7 M dexamethasone in 15-ml polypropylene tubes ("aggregate cultures"). Adipogenic potential was quantitated by flow cytometry of Nile Red-stained cells cultured for 1 and 2 weeks in medium containing isobutyl methylxanthine, indomethacin, insulin, and dexamethasone. Support of osteoclast formation was measured by quantitating multinucleated tartrate-resistant acid phosphatase-positive cells in spleen cell cocultures of test clones (immortomouse clones and positive control ST2 cells) cultured in the presence of 10-7 M vitamin D3 and 150 mM ascorbate-2-phosphate. In vivo osteogenic potential was assayed by histologic examination of bone formation in subcutaneous implants, into athymic mouse hosts, of a composite of cells combined with porous calcium phosphate ceramics. The bone marrow-derived clone BMC9 has the potential to express each of the four mesenchymal characteristics tested, while brain fibroblasts, tested under identical conditions, did not exhibit any of these four mesenchymal characteristics. BMC10 cells exhibited osteogenic and chondrogenic phenotypes, but showed only minimal expression of adipocytic or osteoclast-supportive phenotypes. Clone BMC9 is, minimally, a quadripotential MPC isolated from the marrow of an adult mouse that can differentiate into cartilage and adipose, support osteoclast formation, and form bone. The BMC9 clone is an example of an adult-derived multipotential progenitor cell that is situated early in the mesenchymal lineage.

Hyaluronic acid-based polymers as cell carriers for tissue-engineered repair of bone and cartilage.

Culture-expanded bone marrow-derived mesenchymal progenitor cells differentiate into chondrocytes or osteoblasts when implanted subcutaneously in vivo in combination with an appropriate delivery vehicle. This in vivo implantation technique is used to test new materials as putative delivery vehicles in skeletal tissue-engineering models. HYAFF 11 and ACP sponges, two biomaterials based on hyaluronic acid modified by esterification of the carboxyl groups of the glucuronic acid, were tested as osteogenic or chondrogenic delivery vehicles for rabbit mesenchymal progenitor cells and compared with a well characterized porous calcium phosphate ceramic delivery vehicle. The implant materials were examined by scanning electron microscopy for differences in pore structure or cellular interactions, were quantified for their ability to bind and retain mesenchymal progenitor cells, and were examined histologically for their ability to support osteogenesis and chondrogenesis after subcutaneous implantation into nude mice. The ACP sponge bound the same number of cells as fibronectin-coated ceramic, whereas the HYAFF 11 sponge bound 90% more. When coated with fibronectin, ACP and HYAFF 11 bound, respectively, 100 and 130% more cells than the coated ceramics. HYAFF 11 sponge composites retained their integrity after the 3 or 6-week incubation period in the animals and were processed for histomorphometric analysis. As a result of rapid degradation or resorption in vivo, ACP sponges could not be recovered after implantation and could not be analyzed. HYAFF 11 sponges presented more area available for cell attachment and more available volume for newly formed tissue. Following loading with mesenchymal progenitor cells and implantation, the pores of the sponges contained more bone and cartilage than the pores of ceramic cubes at either time point. Thus, relative to ceramic, HYAFF 11 sponges allow incorporation of twice as many cells and produce a 30% increase in the relative amount of bone and cartilage per unit area. Hence, the hyaluronic acid-based delivery vehicles are superior to porous calcium phosphate ceramic with respect to the number of cells loaded per unit volume of implant, and HYAFF 11 sponges are superior to the ceramics with regard to the amount of bone and cartilage formed. Additionally, hyaluronic acid-based vehicles have the advantage of degradation/resorption characteristics that allow complete replacement of the implant with newly formed tissue.

Vascular smooth muscle differentiation of murine stroma: a sequential model.

Previous studies by our group showed that stromal cells from human long-term marrow cultures were mesenchymal cells following a vascular smooth muscle pathway. The present study using 58 immortalized stromal lines from different hematopoietic sites was conducted to verify whether this hypothesis also held true for murine stroma. Principal components analysis performed using cytoskeletal and extracellular matrix proteins allowed the segregation of five factors explaining more than 70% of the variance. Factor I, including osteopontin and vimentin, and factor II, laminins and fibronectins, were representative of the mesenchyme. The remaining three factors were representative of vascular smooth muscle: factor III, including alphaSM actin, SM alpha actinin, SM22alpha, EDa+ fibronectin, and thrombospondin-1; factor IV, metavinculin and h-caldesmon; and factor V, smooth muscle myosin SM1 and desmin. All lines expressed factors I and II; 53 lines expressed factor III, 35 lines expressed factor IV; and 11 lines expressed factor V. A second principal components analysis including membrane antigens indicated the cosegregration of vascular cell adhesion molecule-1 with osteopontin and that of Ly6A/E with vimentin, whereas CD34 and Thy-1 appeared to be independent factors. The heterogeneity of vascular smooth muscle markers expression suggests that harmonious maintenance of hematopoiesis depends on the cooperation between different stromal cell clones.

In vivo osteogenesis assay: a rapid method for quantitative analysis.

A quantitative in vivo osteogenesis assay is a useful tool for the analysis of cells and bioactive factors that affect the amount or rate of bone formation. There are currently two assays in general use for the in vivo assessment of osteogenesis by isolated cells: diffusion chambers and porous calcium phosphate ceramics. Due to the relative ease of specimen preparation and reproducibility of results, the porous ceramic assay was chosen for the development of a rapid method for quantitating in vivo bone formation. The ceramic cube implantation technique consists of combining osteogenic cells with 27-mm3 porous calcium phosphate ceramics, implanting the cell-ceramic composites subcutaneously into an immuno-tolerant host, and, after 2-6 weeks, harvesting and preparing the ceramic implants for histologic analysis. A drawback to the analysis of bone formation within these porous ceramics is that the entire cube must be examined to find small foci of bone present in some samples; a single cross-sectional area is not representative. For this reason, image analysis of serial sections from ceramics is often prohibitively time-consuming. Two alternative scoring methodologies were tested and compared to bone volume measurements obtained by image analysis. The two subjective scoring methods were: (1) Bone Scale: the amount of bone within pores of the ceramic implant is estimated on a scale of 0-4 based on the degree of bone fill (0=no bone, 1=up to 25%, 2=25 to 75%, 4=75 to 100% fill); and (2) Percentage Bone: the amount of bone is estimated by determining the percentage of ceramic pores which contain bone. Every tenth section of serially sectioned cubes was scored by each of these methods under double-blind conditions, and the Bone Scale and Percentage Bone results were directly compared to image analysis measurements from identical samples. Correlation coefficients indicate that the Percentage Bone method was more accurate than the Bone Scale scoring method. The Bone Scale scoring method gave an r2=0.767 while the Percentage Bone method gave a value of 0.902. These results indicate that scoring ceramic cubes by the percentage of pores containing bone gives a result that corresponds to image analysis measurements at nearly a 90% confidence level. Thus, the Percentage Bone method of scoring is an accurate and relatively quick scoring method for in vivo bone formation.

Amendments to the Center for Devices and Radiological Health federal performance standard for laser products.

Federal law requires that all laser products that are imported into or introduced into commerce in the United States comply with the performance standard published in the Code of Federal Regulations (CRF), Title 21, Parts 1040.10 and 1040.11, administered by the Center for Devices and Radiological Health (CDRH), US Food and Drug Administration. Although it contains somewhat different requirements for hazard classification, engineering controls and labeling, the ANSI Z136.1 standard defers to the CDRH standard. The CDRH standard became effective in August, 1976 and was amended, in 1978 and also in 1985. In the early 1990s, US experts met to formulate an approach to bring the requirements of the CDRH standard and those of the International Electrotechnical Commission (IEC) standard, IEC 825, into closer agreement in order to lower barriers to international trade and to remove any excessive compliance burdens on manufacturers. In 1993, the CDRH published, formally in the Federal Register and informally, a Notice of Intent to amend the CDRH standard. Responses to those notices have now been analyzed and informal draft amendments were distributed in 1996. This draft is now being prepared for formal issuance as a Notice of Proposed Rulemaking. Meanwhile, the IEC standard was amended in 1993 and republished as IEC 825-1; these amendments created considerable controversy since they resulted in over classification of the hazard of many products, especially light emitting diodes (LEDs) that have a large divergence and increased source dimensions. Additional amendments are now being developed to correct this problem. The CDRH has carefully monitored developments in the IEC and actively participated in its proceedings as a guide in developing its own proposal. This paper describes the major changes that are being proposed for the CDRH standard and presents some rationale for the major changes. The more significant changes include expansion of applicability to include LEDs, reduced emission durations for classification, revised measurement for hazard classification, reduced performance requirements for lower power visible radiation products, and revised requirements for medical products.

Alterations in sarcomere structure, collagen organization, mitochondrial activity, and protein metabolism in the avian low score normal muscle weakness.

Skeletal muscle fibers are surrounded by an extracellular matrix. The extracellular matrix is composed of glycoproteins, collagen, and proteoglycans. Proteoglycans have been suggested to play an important functional role in tissue differentiation. However, an understanding of how the extracellular matrix affects skeletal muscle development and function is largely unknown. In the avian genetic muscle weakness, low score normal (LSN), a late embryonic increase in the expression of decorin is followed by a subsequent increase in collagen crosslinking. The sarcomere organization, collagen fibril diameter and organization were investigated using transmission electron microscopy. Measurements were made at 20 days of embryonic development and 6 weeks posthatch. These studies showed changes in sarcomere organization and deterioration of muscle fibril structure in the LSN pectoral muscle. In vitro satellite cell cultures were developed and assayed for mitochondrial activity, and protein synthesis and degradation. In these analyses, mitochondrial activity from LSN satellite cells was significantly higher than those from normal pectoral muscle satellite cells. Protein synthesis rates between the normal and LSN satellite cell-derived myotubes were similar, but protein degradation rates were higher in the LSN cultures. Based on the reported functions of decorin as a regulator of cell proliferation and collagen fibril organization, it is possible that the late embryonic increase in decorin may be influencing the alterations in LSN sarcomere and collagen organization.

Stimulatory effects of basic fibroblast growth factor and bone morphogenetic protein-2 on osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells.

Bone marrow stroma contains multipotential mesenchymal progenitor cells which can differentiate into osteoblastic cells; we refer to these cells as mesenchymal stem cells (MSCs). Basic fibroblast growth factor (bFGF) and bone morphogenetic protein-2 (BMP-2) have been implicated in the osteogenic regulatory process by virtue of their mitogenic and differentiation activities, respectively. This study examines and compares the effects of bFGF and BMP-2 on dexamethasone (Dex)-dependent in vitro osteogenic differentiation of rat marrow-derived MSCs. A 6-day exposure to bFGF markedly stimulated cell growth and induced osteoblastic differentiation as shown by osteocalcin mRNA expression (day 14), bone nodule formation (day 18), and calcium deposition (day 18). These results indicate that bFGF enhances both mitogenic activity and osteogenic development of Dex-treated marrow MSCs. In contrast, BMP-2 did not induce osteogenesis as strongly as bFGF. Thus, exposure to BMP-2 slightly increased bone nodule number and calcium content compared with the control. Exposure of MSCs to both BMP-2 and bFGF induced expression of osteocalcin mRNA and mineralizing bone-like nodules as early as day 11 and resulted in enhancement of bone formation more markedly than either factor alone. Consistent with these results, porous calcium phosphate ceramic cubes implanted in vivo, which were loaded with MSCs pre-exposed to both bFGF and BMP-2, showed higher histologic score for bone formation than those with MSCs pre-exposed to either bFGF or BMP-2 alone. These data indicate that combined treatment with bFGF and BMP-2 synergistically enhances the osteogenic potency of bFGF in rat marrow MSC culture.

LacZ and interleukin-3 expression in vivo after retroviral transduction of marrow-derived human osteogenic mesenchymal progenitors.

Human marrow-derived mesenchymal progenitor cells (hMPCs), which have the capacity for osteogenic and marrow stromal differentiation, were transduced with the myeloproliferative sarcoma virus (MPSV)-based retrovirus, vM5LacZ, that contains the LacZ and neo genes. Stable transduction and gene expression occurred in 18% of cells. After culture expansion and selection in G418, approximately 70% of neo(r) hMPCs co-expressed LacZ. G418-selected hMPC retain their osteogenic potential and form bone in vivo when seeded into porous calcium phosphate ceramic cubes implanted subcutaneously into SCID mice. LacZ expression was evident within osteoblasts and osteocytes in bone developing within the ceramics 6 and 9 weeks after implantation. Likewise, hMPCs transduced with human interleukin-3 (hIL-3) cDNA, adhered to ceramic cubes and implanted into SCID mice, formed bone and secreted detectable levels of hIL-3 into the systemic circulation for at least 12 weeks. These data indicate that genetically transduced, culture-expanded bone marrow-derived hMPCs retain a precursor phenotype and maintain similar levels of transgene expression during osteogenic lineage commitment and differentiation in vivo. Because MPCs have been shown to differentiate into bone, cartilage, and tendon, these cells may be a useful target for gene therapy.

Osteochondrogenic potential of marrow mesenchymal progenitor cells exposed to TGF-beta 1 or PDGF-BB as assayed in vivo and in vitro.

Mesenchymal progenitors cells can be isolated from rat bone marrow and mitotically expanded in vitro. When these cells, which we operationally call mesenchymal stem cells (MSCs), are placed in an appropriate environment, they have the capacity to differentiate into bone and/or cartilage. This capacity is called osteochondrogenic potential. In this study, preconfluent MSCs were exposed in vitro to 5 ng/ml transforming growth factor-beta 1 (TGF-beta 1) or platelet-derived growth factor, isoform BB (PDGF-BB) for a pulse of 48 h and assayed for cell proliferation, alkaline phosphatase activity, and osteochondrogenic potential; untreated MSC's served as controls. In these cell culture conditions, TGF-beta 1 or PDGF-BB had similar effects on proliferation and alkaline phosphatase activity. Both growth factors increased cell proliferation and decreased alkaline phosphatase activity of MSCs. Sister cultures of TGF-beta 1- or PDGF-BB-treated MSCs and untreated MSCs were trypsinized. For each type of culture, the trypsinised MSCs were split in two parts: one part was replated in an osteogenic medium to assess its in vitro osteogenic potential, whereas the other part was seeded into porous calcium phosphate ceramics and implanted subcutaneously in syngeneic rats to assess its in vivo osteochondrogenic potential. PDGF-pretreated MSCs showed no difference in in vivo and in vitro osteochondrogenesis from that of control MSCs, while TGF-beta 1 pretreatment blocked the osteochondrogenic potential of MSCs when assayed in vitro for bone nodule formation. However, when tested in vivo, TGF-beta 1-pretreated MSCs were able to form bone and cartilage. These data show that measurements of proliferation and alkaline phosphatase activity of preconfluent MSCs immediately after exposure to growth factor were not predictive of their subsequent osteochondrogenic potential. Moreover, the variation of the osteochondrogenic potential of MSCs after exposure to growth factor was further modulated by the environment in which the MSCs were assayed.

Differentiation potential of conditionally immortalized mesenchymal progenitor cells from adult marrow of a H-2Kb-tsA58 transgenic mouse.

Primary cultures were initiated from marrow, spleen, and bone explants of an adult H-2Kb-tsA58 transgenic mouse (immortomouse). All cultures were initiated in immortalizing conditions, and an additional marrow culture was first incubated for 1 week in standard conditions and then switched to immortalizing conditions. Marrow cells immediately immortalized were designated the marrow immediate population (MIP); those immortalized after 1 week were termed the marrow delayed population (MDP). MIP and MDP cells both contained a mixture of fibroblastic or flattened cells, and the MIP cells contained an additional subpopulation of adipocytic (Oil Red-O positive) cells. Alkaline phosphatase expression was induced by dexamethasone (10(-7) M) in MDP cells while MIP, spleen, and bone explant cells had only a low level of expression. MDP and MIP cells differentiated into bone when combined with porous calcium phosphate ceramics and implanted subcutaneously into nude mice while bone- and spleen-derived cells did not. Clones were isolated from the MDP and MIP cell populations and tested for differentiated phenotypes. Some MIP-derived clones exhibited adipocytic characteristics while MDP-derived subclones were negative. Histologic examination of porous ceramic implanted clones showed that all of the clones had osteogenic potential. Clones exposed to either dexamethasone, human recombinant bone morphogenetic protein-2, or horse serum plus hydrocortisone showed differences in expression of adipocytic or osteogenic markers. These immortalized cultures have retained both adipocytic and osteogenic potential even after 1 year of continuous culture, and provide a model system for clonal analysis of the developmental potential of marrow-derived mesenchymal precursor cells.