Soumya Ravindran

Changes of chondrocyte expression profiles in human MSC aggregates in the presence of PEG microspheres and TGF-β3

Biomaterial microparticles are commonly utilized as growth factor delivery vehicles to induce chondrogenic differentiation of mesenchymal stem/stromal cells (MSCs). To address whether the presence of microparticles could themselves affect differentiation of MSCs, a 3D co-aggregate system was developed containing an equal volume of human primary bone marrow-derived MSCs and non-degradable RGD-conjugated poly(ethylene glycol) microspheres (PEG-μs). Following TGF-β3 induction, differences in cell phenotype, gene expression and protein localization patterns were found when compared to MSC aggregate cultures devoid of PEG-μs. An outer fibrous layer always found in differentiated MSC aggregate cultures was not formed in the presence of PEG-μs. Type II collagen protein was synthesized by cells in both culture systems, although increased levels of the long (embryonic) procollagen isoforms were found in MSC/PEG-μs aggregates. Ubiquitous deposition of type I and type X collagen proteins was found in MSC/PEG-μs cultures while the expression patterns of these collagens was restricted to specific areas in MSC aggregates. These findings show that MSCs respond differently to TGF-β3 when in a PEG-μs environment due to effects of cell dilution, altered growth factor diffusion and/or cellular interactions with the microspheres. Although not all of the expression patterns pointed toward improved chondrogenic differentiation in the MSC/PEG-μs cultures, the surprisingly large impact of the microparticles themselves should be considered when designing drug delivery/scaffold strategies.

Molecular properties and fibril ultrastructure of types II and XI collagens in cartilage of mice expressing exclusively the α1(IIA) collagen isoform

Until now, no biological tools have been available to determine if a cross-linked collagen fibrillar network derived entirely from type IIA procollagen isoforms, can form in the extracellular matrix (ECM) of cartilage. Recently, homozygous knock-in transgenic mice (Col2a1(+ex2), ki/ki) were generated that exclusively express the IIA procollagen isoform during post-natal development while type IIB procollagen, normally present in the ECM of wild type mice, is absent. The difference between these Col2a1 isoforms is the inclusion (IIA) or exclusion (IIB) of exon 2 that is alternatively spliced in a developmentally regulated manner. Specifically, chondroprogenitor cells synthesize predominantly IIA mRNA isoforms while differentiated chondrocytes produce mainly IIB mRNA isoforms. Recent characterization of the Col2a1(+ex2) mice has surprisingly shown that disruption of alternative splicing does not affect overt cartilage formation. In the present study, biochemical analyses showed that type IIA collagen extracted from ki/ki mouse rib cartilage can form homopolymers that are stabilized predominantly by hydroxylysyl pyridinoline (HP) cross-links at levels that differed from wild type rib cartilage. The findings indicate that mature type II collagen derived exclusively from type IIA procollagen molecules can form hetero-fibrils with type XI collagen and contribute to cartilage structure and function. Heteropolymers with type XI collagen also formed. Electron microscopy revealed mainly thin type IIA collagen fibrils in ki/ki mouse rib cartilage. Immunoprecipitation and mass spectrometry of purified type XI collagen revealed a heterotrimeric molecular composition of α1(XI)α2(XI)α1(IIA) chains where the α1(IIA) chain is the IIA form of the α3(XI) chain. Since the N-propeptide of type XI collagen regulates type II collagen fibril diameter in cartilage, the retention of the exon 2-encoded IIA globular domain would structurally alter the N-propeptide of type XI collagen. This structural change may subsequently affect the regulatory function of type XI collagen resulting in the collagen fibril and cross-linking differences observed in this study.

Monoclonal antibodies to intracellular stages of Cryptosporidium parvum define life cycle progression in vitro

Cryptosporidium is a protozoan parasite that causes gastrointestinal disease in humans and animals. Currently, there is a limited array of antibodies available against the parasite, which hinders imaging studies and makes it difficult to visualize the parasite life cycle in different culture systems. In order to alleviate this reagent gap, we created a library of novel antibodies against the intracellular life cycle stages of Cryptosporidium. We identified antibodies that recognize specific life cycle stages in distinctive ways, enabling unambiguous description of the parasite life cycle. These MAbs will aid future investigation into Cryptosporidium biology and help illuminate growth differences between various culture platforms. ABSTRACT Among the obstacles hindering Cryptosporidium research is the lack of an in vitro culture system that supports complete life development and propagation. This major barrier has led to a shortage of widely available anti-Cryptosporidium antibodies and a lack of markers for staging developmental progression. Previously developed antibodies against Cryptosporidium were raised against extracellular stages or recombinant proteins, leading to antibodies with limited reactivity across the parasite life cycle. Here we sought to create antibodies that recognize novel epitopes that could be used to define intracellular development. We identified a mouse epithelial cell line that supported C. parvum growth, enabling immunization of mice with infected cells to create a bank of monoclonal antibodies (MAbs) against intracellular parasite stages while avoiding the development of host-specific antibodies. From this bank, we identified 12 antibodies with a range of reactivities across the parasite life cycle. Importantly, we identified specific MAbs that can distinguish different life cycle stages, such as trophozoites, merozoites, type I versus II meronts, and macrogamonts. These MAbs provide valuable tools for the Cryptosporidium research community and will facilitate future investigation into parasite biology. IMPORTANCE Cryptosporidium is a protozoan parasite that causes gastrointestinal disease in humans and animals. Currently, there is a limited array of antibodies available against the parasite, which hinders imaging studies and makes it difficult to visualize the parasite life cycle in different culture systems. In order to alleviate this reagent gap, we created a library of novel antibodies against the intracellular life cycle stages of Cryptosporidium. We identified antibodies that recognize specific life cycle stages in distinctive ways, enabling unambiguous description of the parasite life cycle. These MAbs will aid future investigation into Cryptosporidium biology and help illuminate growth differences between various culture platforms.

75 GENERATION OF A TRANSGENIC KNOCK-IN MOUSE MODEL EXPRESSING ONLY THE IIA ISOFORM OF TYPE II PROCOLLAGEN

Purpose: To generate a mouse model where alternative pre-mRNA splicing of the type II procollagen gene (COL2A1) is inhibited. Normally, chondroprogenitor cells synthesize predominantly exon 2-containing mRNA isoforms of COL2A1 (type IIA and IID) while differentiated chondrocytes mainly generate COL2A1 mRNA isoforms devoid of exon 2 (type IIB). The biological significance of this splicing switch with respect to cartilage/skeletal development and maintenance is not known. To address this issue, we synthesized a transgenic mouse expressing predominantly the IIA isoform of COL2A1 by altering the 5′ splice site sequence of exon 2. Methods: A 4ntd mutation was created at the 5′ splice site of exon 2 in a COL2A1 mini-gene construct. This mutation converts exon 2 from a weak splice site to strong consensus splice site. Wild type and mutant minigenes were transfected into chondrocyte cell lines and spliced isoforms were analyzed by RT-PCR. To generate the mouse model, a targeting vector was made containing one short arm (~2kB) and one long arm (~7kb including the 4ntd splice site mutation) of the Col2a1 locus. Targeting vector was electroporated into ES cells (129S6/SvEvTac) and Southern blotting identified homologously-recombined clones. Positive ES clones were injected into C57BL/6 blastocysts to generate chimeric mice. Further breedings were done to generate heterozygote mice devoid of the neomycin cassette by crossing to Cre-expressing mice (EIIa-Cre). Resulting heterozygote mice were bred to generate WT, +/− and −/− mice for analyses. Epiphyseal cartilage was dissected from limbs of P7, P14 and P28 litters and RT-PCR was carried out to detect the ratio of IIA / IID and IIB mRNA. Paraffin tissue sections of hindlimbs were analyzed by Safranin-O staining or immunohistochemistry using antibodies against the triple-helical domain or the exon2-encoded domain of type II procollagen. 4M guanidine extracts from epiphyseal cartilage tissue were analyzed by Western blotting using the same type II procollagen Abs. Results: Splicing of the mutant COL2A1 mini-gene by cells in vitro resulted in synthesis of predominantly the IIA isoform. Homologous recombination of the targeting vector in vivo resulted in positive ES clones. Chimeric mice were generated following blastocyst injections of ES clones and germline transmission was confirmed by PCR. Homozygote mice were generated at the expected Mendelian ratio. No overt phenotype was noted in the +/− and −/− mice compared to WT littermates. RT-PCR confirmed that IIA is the predominant isoform produced in epiphyseal cartilage from homozygote mice while WT mice produced mainly IIB mRNA at all three time points (P7, P14, P28) as expected. Immunohistochemistry showed a dramatic increase in localization of the IIA propeptide in proximal tibial cartilage of +/− and −/− mice when compared to WT. Western blotting showed that procollagen processing occurs in the matrix of +/− and −/− mice at P7 although levels of unprocessed proa1 (IIA) and pN-a1(IIA) were also identified. Conclusions: Global knock-in mice have been generated that express predominantly the IIA isoform of type II procollagen by altering a 4ntd sequence of the 5′ splice site of exon 2. Preliminary histological analyses suggest that cartilage development is normal even though there is persistent and high expression of IIA propeptide protein in the matrix at post-natal time points when this protein is not normally present. Future studies will involve analyzing whether other cartilage matrix components have been altered as a result of changing type II collagen isoform expression. Investigation of cartilage maintenance over time with normal ageing or as a result of osteoarthritis induction (DMM model) will also be carried out.

Assessment of epiphyseal plate allograft viability and function after ex vivo storage in University of Wisconsin Solution.

Background: Compromised epiphyseal plate function can result in limb deformities. Microvascular transplantation of an epiphyseal plate allograft is a potentially effective approach to reestablish longitudinal limb growth. For this procedure to become clinically useful, the technique for temporary ex vivo storage of allografts must be reliable. The goal of this study was to determine a time frame for which proximal tibial epiphyseal plate allografts could be stored in University of Wisconsin Preservation Solution (UWPS) and remain functional in vivo after microvascular transplantation. Methods: Proximal tibial epiphyseal plate allografts from skeletally immature female New Zealand White rabbits (10 to 12 wk of age) were used. Allografts (isolated on the popliteal arteriovenous pedicle) were stored ex vivo in cold UWPS for periods of up to 21 days. Chondrocyte viability, phenotype, and extracellular matrix composition of growth plate cartilage was assessed. Microvascular transplantations of nonstored or prestored (3 d) allografts were performed and analysis of bromodeoxyuridine and calcein incorporation was done to determine chondrocyte proliferation and new bone growth, respectively. Results: In vitro analysis showed that, compared with control tissue, epiphyseal plate chondrocyte viability (P>0.05), organization, and collagen extracellular matrix was preserved up to 4 days in cold UWPS. Microvascular transplantation of nonstored epiphyseal plate allografts was successful. Despite care being taken to ensure vascular patency during the microvascular procedure, transplantation of prestored allografts failed due to absent flow in the larger vessels and in the allograft based upon the visualization of organized thrombus within the vascular pedicle, and absent flow within the composite graft itself. However, growth plate viability and function was detected in a peripheral region of a single allograft where partial blood flow had been maintained during the transplantation period. Conclusions: Ex vivo storage in cold UWPS for 3 days maintains growth plate chondrocyte viability and function in vivo. However, future studies must be directed toward investigating the direct effect of ex vivo storage on the integrity and function of the vascular pedicles.

Microvascular Transplantation of Epiphyseal Plate Allografts Following ex vivo Storage in University of Wisconsin Preservation Solution: Not a clinical study

INTRODUCTION An effective strategy to address issues of epiphyseal growth plate dysfunction would be the microvascular transplantation of a donor epiphyseal plate allograft. If clinical utilization of cadaveric allografts is to be considered, then a reliable procedure for temporary storage that preserves cellular viability and function must be demonstrated. The goal of this study is to determine a suitable time frame that donor epiphyseal plate allografts can be stored ex vivo in University of Wisconsin Storage Solution (UWSS) such that functional biological activity will be maintained in vivo. Our data indicate that storage of rabbit proximal tibial epiphyseal growth plate allografts for at least 3 days in UWSS preserves chondrocyte viability, proliferation and biosynthetic activity to permit new bone growth in vivo following short-term microvascular transplantation.