Biomedical subjects

Gloria Gronowicz

Publications and source records attributed to Gloria Gronowicz.

8 recordsLinked to original sources

PubMed2006

Matrix-mediated retention of in vitro osteogenic differentiation potential and in vivo bone-forming capacity by human adult bone marrow-derived mesenchymal stem cells during ex vivo expansion.

Mesenchymal stem cells (MSCs) represent an attractive cell source for tissue engineering applications, since they are readily isolated from adult bone marrow and have the ability to differentiate along multiple mesenchymal lineages, including osteogenic. Currently, utilization of MSCs for bone tissue engineering is limited because of the attenuation of their osteogenic differentiation potential and in vivo bone-forming capacity following ex vivo expansion on conventional tissue culture plastic (TCP). Previously, we demonstrated that a denatured type I collagen (DC) matrix promotes the maintenance of MSC in vitro osteogenic differentiation potential during ex vivo expansion in contrast to TCP. In this study, we further demonstrate that the maintenance of MSC osteogenic differentiation potential is primarily due to the ability of DC matrix to influence the retention of early passage osteogenic functions in late passage (LP) cells during ex vivo expansion, in contrast to solely enhancing attenuated LP cellular functions during osteogenic differentiation. Serum-associated factors played a significant role in influencing the retention of MSC osteogenic differentiation potential during expansion on the DC matrix. Significantly, the results show that although LP cells expanded ex vivo on TCP highly attentuate their in vivo bone-forming capacity, the expansion of MSCs on DC matrix preserves this ability as determined by histological, histomorphometric, and bone mineral density evaluations of MSC-seeded hydroxyapatite/tricalcium phosphate scaffolds following an 8-week implantation period within a heterotopic muscle pouch model. These findings provide further insight into the importance of matrix-mediated effects on MSC function and selective factors important in this process.

Adult Stem Cells↗

PubMed2006

Interleukin-7 influences osteoclast function in vivo but is not a critical factor in ovariectomy-induced bone loss.

UNLABELLED: IL-7 is produced by stromal cells in bone marrow and is a major regulator of B and T lymphopoiesis. It is also a direct inhibitor of osteoclastogenesis in vitro. In this study we show that IL-7-deficient mice have increased OC and decreased trabecular bone volume compared with WT mice but mimic WT mice in the amount of trabecular but not cortical bone lost after ovariectomy. INTRODUCTION: Interleukin (IL)-7 is a potent regulator of lymphocyte development, which has significant effects on bone. Bone marrow cell cultures from IL-7 deficient (IL-7KO) mice produced significantly more TRACP(+) osteoclasts (OCs) than did cells from wildtype (WT) mice. A previous study found that treatment of mice with a neutralizing antibody to IL-7 blocked ovariectomy (OVX)-induced bone loss. We examined if differences exist between the bones of WT and IL-7KO mice and if OVX altered bone mass in IL-7KO mice. MATERIALS AND METHODS: Studies were in 2-month-old sham-operated (SHAM) and OVX female mice that were killed 4 weeks after surgery. IL-7KO mice and WT controls were in a C57BL/6 background. Both vertebrae (L(1)) and femora were evaluated by DXA, muCT, and histomorphometry. IL-7KO mice were confirmed as IL-7 deficient by their almost total lack of mature B cells in their bone marrow. RESULTS: There was significantly less trabecular bone volume in the vertebrae of IL-7KO mice than in WT mice. In addition, IL-7KO mice had significantly decreased (p < 0.05) trabecular number (13%) and increased trabecular spacing (15%). OVX decreased vertebral trabecular bone volume (TBV) by 21% (p < 0.05) in WT mice and by 22% (p < 0.05) in IL-7KO mice compared with SHAM. IL-7KO SHAM mice also had significantly less (30%) TBV (TA/TTA) in their femurs, as measured histomorphometrically, than did WT SHAM mice. Femurs from IL-7KO SHAM mice had significantly increased percent OC surface (23%) compared with WT SHAM. As in the vertebrae, OVX significantly decreased femoral TBV in both WT and IL-7KO mice by similar amounts (47% and 48%, respectively, p < 0.05 for both) compared with SHAM. However, OVX decreased cortical bone mass in WT but not in IL-7KO bones. We also examined bone marrow cells from WT and IL-7KO mice. Bone marrow cells from IL-7KO animals showed a significant increase in the number of TRACP(+) osteoclast-like cells (OCLs), which formed in cultures that were stimulated with macrophage-colony stimulating factor (M-CSF) and RANKL (both at 30 ng/ml). However, there was no significant difference in the number of OCLs that formed in B lymphocyte-depleted (B220(-)) bone marrow cell cultures from WT and IL-7KO mice. CONCLUSIONS: IL-7 deficiency in mice caused increased OC number in bone and decreased bone mass. OVX-induced bone loss in IL-7-deficient mice was selective and occurred in trabecular but not cortical bone.

Absorptiometry, Photon↗

PubMed2006

T lymphocyte-deficient mice lose trabecular bone mass with ovariectomy.

UNLABELLED: We examined OVX-induced bone loss in three TLD mouse models. In TLD mice, OVX caused trabecular bone loss equivalent to that of WT. In contrast, cortical bone loss with OVX was variable. We conclude that T lymphocytes do not influence OVX-induced trabecular bone loss. INTRODUCTION: We examined ovariectomy (OVX)-induced bone loss in three T lymphocyte-deficient (TLD) mouse models: nude mice, recombination activating gene 2-deficient (RAG2 KO) mice, and T cell receptor alpha chain-deficient (TCRalpha KO) mice. MATERIALS AND METHODS: Bone mass was examined by DXA, microCT, and histomorphometry. We also examined the effect of OVX on T lymphocytes in the bone marrow and spleens of wildtype (WT) mice and on in vitro osteoclastogenesis and colony forming unit-granulocyte macrophage (CFU-GM) activity in the bone marrow of WT and nude mice. RESULTS: In WT mice, OVX did not alter T lymphocyte number in the bone marrow but did increase T lymphocytes in the spleen. Comparison of bone mass in nude, RAG2 KO, and TCRalpha KO mice with WT as measured by DXA showed decreased femoral bone mass in nude mice and increased vertebral bone mass in RAG2 KO mice. In TCRalpha KO mice, femoral, tibial, and vertebral bone mass were decreased. In vertebrae and long bones, bone loss with OVX was consistently present in WT mice but variably present in TLD mice as measured by DXA. In contrast, microCT and histomorphometry showed similar trabecular bone loss after OVX in all mice. However, femoral cortical bone loss occurred only in WT and RAG2 KO mice. OVX produced similar trabecular bone loss in WT and TCRalpha KO mice and also induced cortical bone loss in both. Histomorphometry showed that TRACP(+) area in bones was increased by OVX in femurs from both WT and nude mice as was in vitro osteoclast-like cell formation and CFU-GM activity. CONCLUSIONS: These results show that OVX caused similar trabecular bone loss in both WT and TLD mice. The ability of DXA and measurement of cortical bone loss to show OVX-induced effects on bone mass was variable. It seems that T lymphocytes are not critical for OVX-induced trabecular bone loss in these mouse models.

Animals↗

PubMed2005

The inflammatory responses to silk films in vitro and in vivo.

Silks have a long history of biomedical use as sutures. Silk can be purified, chemically modified to attach RGD sequences and processed into highly porous scaffolds for tissue engineering. We report biocompatibility studies of silk films (with or without covalently bound RGD) that were seeded with bone-marrow derived mesenchymal stem cells (MSC) and (a) cultured in vitro with human MSC or (b) seeded with autologous rat MSC and implanted in vivo. Controls for in vitro studies included tissue culture plastic (TCP; negative control), TCP with lipopolysaccharide (LPS) in the cell culture medium (positive control), and collagen films; controls for in vivo studies included collagen, PLA and TCP. After 9 h of culture, the expression of the pro-inflammatory Interleukin 1 beta (IL-1beta) and inflammatory cyclooxygenase 2 (COX-2) in human MSC were comparable for silk, collagen and TCP. After 30 and 96 h, gene expression of IL-1beta and COX-2 in MSC returned to the baseline (pre-seeding) levels. These data were corroborated by measuring IL-1beta and prostaglandin E2 levels in culture medium. The rate of cell proliferation was higher on silk films than either on collagen or TCP. In vivo, films made of silk, collagen or PLA were seeded with rat MSCs, implanted intramuscularly in rats and harvested after 6 weeks. Histological and immunohistochemical evaluation of silk explants revealed the presence of circumferentially oriented fibroblasts, few blood vessels, macrophages at the implant-host interface, and the absence of giant cells. Inflammatory tissue reaction was more conspicuous around collagen films and even more around PLA films when compared to silk. These data suggest that (a) purified degradable silk is biocompatible and (b) the in vitro cell culture model (hMSC seeded and cultured on biomaterial films) gave inflammatory responses that were comparable to those observed in vivo.

Animals↗

PubMed2005

Effect of platelet-rich plasma with autogenous bone graft for maxillary sinus augmentation in a rabbit model.

PURPOSE: To evaluate the effect of platelet-rich plasma (PRP) on autogenous bone graft remodeling during sinus augmentation in a rabbit model. MATERIALS: Twelve New Zealand White rabbits were divided randomly into 3 groups based on their time of sacrifice (2, 4, and 8 weeks). All animals underwent a general anesthetic and harvesting of an autogenous bone graft from the right iliac crest with subsequent bilateral maxillary sinus augmentation. PRP was prepared via standard approved technique by acquiring 21 cc of autogenous blood and performing differential centrifugation to obtain PRP. One cc of PRP was produced that was mixed with bovine topical thrombin and calcium chloride. The left maxillary sinus received only autogenous bone, while the right maxillary sinus received a mixture of PRP mixed with autogenous bone, thus each animal acted as its own control. Equal volumes of bone were inserted in each maxillary sinus. Animals were sacrificed at 2, 4, and 8 weeks and all specimens were harvested for peripheral quantitative computed tomography (pQ-CT), static, and dynamic and histomorphometric analysis. RESULTS: Student t tests were performed comparing bone density via pQ-CT analysis, histomorphometric parameters of total bone area, and bone apposition rate. PRP had no statistically significant effect on bone graft healing in maxillary sinus augmentation when compared using standard pQ-CT, static, and dynamic histologic criteria. CONCLUSION: This study fails to find a direct stimulatory effect of PRP on healing of autogenous bone grafts using pQ-CT, static, and dynamic histomorphometric analyses.

Alveolar Ridge Augmentation↗

PubMed2004

Do cyclooxygenase-2 knockout mice have primary hyperparathyroidism?

The absence of cyclooxygenase-2 (COX-2) activity in vitro reduces differentiation of both bone-forming and bone-resorbing cells. To examine the balance of COX-2 effects on bone in vivo, we studied COX-2 knockout (KO) and wild-type (WT) mice. After weaning, KO mice died 4 times faster than WT mice, consistent with reports of progressive renal failure in KO mice. Among KO mice killed at 4 months of age, some had renal failure with marked secondary hyperparathyroidism, but others appeared healthy. On the assumption that renal failure was not inevitable in COX-2 KO mice and that phenotypic differences might increase with age, we studied KO mice surviving to 10 months of age with serum creatinine levels similar to those of WT mice. In 10-month-old male KO mice, serum calcium and PTH, but not phosphorus, levels were increased compared with those in WT mice. 1,25-Dihydroxyvitamin D(3) levels were markedly elevated in KO mice. Skeletal analysis showed small nonsignificant decreases in cortical bone density by BMD and either an increase (distal femur, by microcomputed tomography) or no difference (distal femur, by static histomorphometry) in trabecular bone density in KO mice. There was a trend toward increased percent osteoblastic and osteoclastic surfaces, and on dynamic histomorphometry, the rates of trabecular bone formation and mineral apposition were increased in KO mice relative to WT mice. Similar trends were observed for most parameters in 10-month-old female COX-2 KO mice. However, rates of trabecular bone formation and mineral apposition were increased in 10-month-old WT females compared with males and did not increase further in female KO mice. These data suggest that COX-2 KO mice with intact renal function have primary hyperparathyroidism, and that effects of increased PTH and 1,25-dihydroxyvitamin D(3) to increase bone turnover may compensate for the absence of COX-2.

Animals↗

PubMed2004

Stat1 controls postnatal bone formation by regulating fibroblast growth factor signaling in osteoblasts.

Activation of the signal transducers and activators of transcription (STAT) pathway is important in fibroblast growth factor (FGF) modulation of chondrocyte proliferation and endochondral bone formation during embryogenesis. However, it is not known if the FGF/STAT signaling pathway is important for postnatal bone formation. To examine this, we have characterized a novel skeletal phenotype in Stat1-/- mice in which we find a significant increase in bone mineral density, bone mineral content, and other parameters of bone growth. The data show that osteoblasts derived from Stat1-/- mice have decreased expression of cell cycle inhibitor p21WAF/CIP and FGF receptor 3, a known negative regulator of chondrocyte proliferation. Interestingly, Stat1-/- osteoblasts showed increased expression of FGF18 in vivo and increased responsiveness to FGF18 in vitro. These results suggest a mechanism for the regulation of the osteoblast in which Stat1 functions not only to directly regulate the cell cycle but also to modify the repertoire of FGF receptor expression from a potentially inhibitory receptor, FGFR3 to a stimulatory receptor such as FGFR1 or FGFR2.

Alkaline Phosphatase↗

PubMed2003

Effects of transforming growth factor-beta 1 (TGF-beta1) on in vitro mineralization of human osteoblasts on implant materials.

An in vitro cell-implant mineralization model system was used to study the effect of transforming growth factor-beta 1(TGF-beta1) on mineralization in human osteoblast cultures. SaOs-2 and primary human osteoblast (HOB) cells were cultured on Tivanium (Ti-6Al-4V) disks. Administration of different concentrations of TGF-beta1 (0.02, 0.01, 0.2, 1.0, 2.0ng/ml) to these cultures demonstrated a biphasic dose response with 0.2ng/ml TGF-beta1 maximally increasing the calcium content compared to control culture. Results with SaOs-2 and HOB cultures were similar. An optimal dose of TGF-beta1 (0.2ng/ml) was provided to the cultures either in one single dose or multiple doses. Continuous administration of 0.2ng/ml TGF-beta1 caused 77% (SaOs-2) and 60% (HOB) increases in calcification compared to the control and 0.2ng/ml single dose groups. Single administration of the accumulative dose at 1.6ng/ml had no significant effect on the calcium content in either cell culture compared to control. Two weeks continuous administration of 0.2ng/ml TGF-beta1 in both cell cultures resulted in significant increases in the expression of bone specific extracellular matrix proteins which included alkaline phosphatase, Type I collagen, and osteocalcin as measured by Northern blot analysis and RT-PCR. At 4 weeks, the mRNA level of Type I collagen was still significantly higher in the TGF-beta1 treatment group compared to control. In conclusion, TGF-beta1 enhances mineralization of HOB on implant materials.

Alkaline Phosphatase↗