A combined cell and growth factor delivery for the repair of a critical size tibia defect using biodegradable hydrogel implants.

The ability to repair critical-sized long-bone injuries using growth factor and cell delivery was investigated using hydrogel biomaterials. Physiological doses of the recombinant human bone morphogenic protein-2 (rhBMP2) were delivered in a sustained manner from a biodegradable hydrogel containing peripheral human blood-derived endothelial progenitor cells (hEPCs). The biodegradable implants made from polyethylene glycol (PEG) and denatured fibrinogen (PEG-fibrinogen, PF) were loaded with 7.7 µg/ml of rhBMP2 and 2.5 × 106 cells/ml hEPCs. The safety and efficacy of the implant were tested in a rodent model of a critical-size long-bone defect. The hydrogel implants were formed ex-situ and placed into defects in the tibia of athymic nude rats and analyzed for bone repair after 13 weeks following surgery. The hydrogels containing a combination of 7.7 µg/ml of rhBMP2 and 2.5 × 106 cells/ml hEPCs were compared to control hydrogels containing 7.7 µg/ml of rhBMP2 only, 2.5 × 106 cells/ml hEPCs only, or bare hydrogels. Assessments of bone repair include histological analysis, bone formation at the site of implantation using quantitative microCT, and assessment of implant degradation. New bone formation was detected in all treated animals, with the highest amounts found in the treatments that included animals that combined the PF implant with rhBMP2. Moreover, statistically significant increases in the tissue mineral density (TMD), trabecular number and trabecular thickness were observed in defects treated with rhBMP2 compared to non-rhBMP2 defects. New bone formation was significantly higher in the hEPC-treated defects compared to bare hydrogel defects, but there were no significant differences in new bone formation, trabecular number, trabecular thickness or TMD at 13 weeks when comparing the rhBMP2 + hEPCs-treated defects to rhBMP2-treated defects. The study concludes that the bone regeneration using hydrogel implants containing hEPCs are overshadowed by enhanced osteogenesis associated with sustained delivery of rhBMP2.

Characterization of Factors Affecting the Detection Limit of EGFR p.T790M in Circulating Tumor DNA.

OBJECTIVE: Circulating tumor DNA is a promising noninvasive tool for cancer monitoring. One of the challenges in applying this tool is the detection of low-frequency mutations. The detection limit of these mutations varies between different molecular methods. The aim of this study is to characterize the factors affecting the limit of detection for epidermal growth factor receptor p.T790M mutation in circulating tumor DNA of patients with lung adenocarcinoma. METHODS: DNA was extracted from plasma samples of 102 patients. For sequencing the DNA, we used 2 different next-generation sequencing-based platforms: Ion Torrent Personal Genome Machine (56 cases) and Roche/454 (46 cases). Serially diluted synthetic DNA samples carrying the p.T790M mutation were sequenced using the Ion Torrent Personal Genome Machine for validation. Limit of detection was determined through the analysis of non-hot-spot nonreference reads, which were regarded as sequencing artifacts. RESULTS: The frequency of the non-hot-spot nonreference reads was higher in Ion Torrent Personal Genome Machine compared to Roche/454 (0.07% ± 0.08% and 0.03% ± 0.06%, respectively, P < .001). We found that different base type substitutions occur with different frequency. Since the base substitution leading to p.T790M mutation is C>T transition, its frequency was used to determine the limit of detection for the assay. Based on the C>T non-hot-spot nonreference allele frequency, we found that the limit of detection is 0.18% in Ion Torrent Personal Genome Machine and 0.1% in Roche/454. Based on these values, 48% and 56% of the cases were positive for T790M mutation in Ion Torrent Personal Genome Machine and Roche/454 groups, respectively. Agreement between duplicates was 76% in Ion Torrent Personal Genome Machine and 72% in Roche/454. Using serially diluted synthetic DNA samples carrying the p.T790M mutation, we could identify mutations with allele frequency of 0.18% or more using the Ion Torrent Personal Genome Machine, supporting our approach to determine the detection limit. CONCLUSION: Both the sequencing platform and the specific nucleotide change affect the limit of detection and should therefore be determined in the validation process of new assays.

Mutant KRAS Circulating Tumor DNA Is an Accurate Tool for Pancreatic Cancer Monitoring.

BACKGROUND: Many new pancreatic cancer treatment combinations have been discovered in recent years, yet the prognosis of pancreatic ductal adenocarcinoma (PDAC) remains grim. The advent of new treatments highlights the need for better monitoring tools for treatment response, to allow a timely switch between different therapeutic regimens. Circulating tumor DNA (ctDNA) is a tool for cancer detection and characterization with growing clinical use. However, currently, ctDNA is not used for monitoring treatment response. The high prevalence of KRAS hotspot mutations in PDAC suggests that mutant KRAS can be an efficient ctDNA marker for PDAC monitoring. SUBJECTS, MATERIALS, AND METHODS: Seventeen metastatic PDAC patients were recruited and serial plasma samples were collected. CtDNA was extracted from the plasma, and KRAS mutation analysis was performed using next-generation sequencing and correlated with serum CA19-9 levels, imaging, and survival. RESULTS: Plasma KRAS mutations were detected in 5/17 (29.4%) patients. KRAS ctDNA detection was associated with shorter survival (8 vs. 37.5 months). Our results show that, in ctDNA positive patients, ctDNA is at least comparable to CA19-9 as a marker for monitoring treatment response. Furthermore, the rate of ctDNA change was inversely correlated with survival. CONCLUSION: Our results confirm that mutant KRAS ctDNA detection in metastatic PDAC patients is a poor prognostic marker. Additionally, we were able to show that mutant KRAS ctDNA analysis can be used to monitor treatment response in PDAC patients and that ctDNA dynamics is associated with survival. We suggest that ctDNA analysis in metastatic PDAC patients is a readily available tool for disease monitoring. IMPLICATIONS FOR PRACTICE: Avoiding futile chemotherapy in metastatic pancreatic ductal adenocarcinoma (PDAC) patients by monitoring response to treatment is of utmost importance. A novel biomarker for monitoring treatment response in PDAC, using mutant KRAS circulating tumor DNA (ctDNA), is proposed. Results, although limited by small sample numbers, suggest that ctDNA can be an effective marker for disease monitoring and that ctDNA level over time is a better predictor of survival than the dynamics of the commonly used biomarker CA19-9. Therefore, ctDNA analysis can be a useful tool for monitoring PDAC treatment response. These results should be further validated in larger sample numbers.

Relationship Between Pityriasis Lichenoides and Mycosis Fungoides: A Clinicopathological, Immunohistochemical, and Molecular Study.

BACKGROUND: Several cases of pityriasis lichenoides (PL) have been reported to evolve into mycosis fungoides (MF). OBJECTIVE: To elucidate clues to this progression. METHODS: Fifty-eight patients with PL between 2000 and 2013 (follow-up: 3-16 years, average: 8.3). RESULTS: A total of 3 (5.2%) of the 58 patients with PL developed MF after 3-11 years of prolonged clinical course. Papules and small plaques characterized PLs, and patches and larger plaques subsequent MFs. A total of 35 of 41 (85%) followed up non-MF associated patients with PL reported lasting complete remissions. Histopathologically, apoptotic keratinocytes disappeared mostly or completely in subsequent MFs. The presence of epidermotropism, folliculotropism, and epidermal lymphocytic nuclear atypia in PLs was not predictive of MF. CD8 cells were the dominant intraepidermal lymphocytes in the 3 PLs but remained so in only 1 subsequent MF. CD7 lymphocytes decreased substantially in 2 MFs, and lymphocytic nuclear atypia increased markedly in 1. T-cell receptor gene rearrangement studies demonstrated clonal populations in 1 of 2 studied PLs and in all 3 subsequent MFs. CONCLUSIONS: A few PLs may evolve into MF. Prolonged clinical course, appearance of patches and larger plaques, markedly increased lymphocytic nuclear atypia, marked diminution of apoptotic keratinocytes and CD7 and CD8 lymphocytes, and clonal T-cell receptor gene rearrangement may serve as clues.

Clinically significant sub-clonality for common drivers can be detected in 26% of KRAS/EGFR mutated lung adenocarcinomas.

Genetic sub-clonality has been described in multiple malignancies, however the presence of sub-clonality for major drivers in lung adenocarcinoma and its clinical significance is a subject under debate. Using molecular and morphometric approach, 347 lung adenocarcinoma samples were analyzed for KRAS and EGFR sub-clonality, which was further correlated with clinical and pathological variables.KRAS and EGFR mutations were identified in 100 (29%) and 82 (23%) cases, respectively. One hundred and forty four KRAS or EGFR positive cases were also available for morphometric analysis, among which 37 (26%) were defined as sub-clonal. The presence of sub-clonality was associated with shorter survival time (p=0.02). Interestingly, cases with sub-clonality were also associated with earlier disease stage (89% vs 66% stage I disease in sub-clonal vs clonal cases, respectively, p=0.01) and less lymph node involvement (8% vs 25% in sub-clonal vs clonal cases, respectively, p=0.02). Our findings demonstrate the presence of sub-clonality for mutations in common drivers in lung adenocarcinoma and link it both to earlier disease stage and to poor survival. These findings are in line with the different evolutionary models that can present with genetic sub-clonality.

Variation in KRAS driver substitution distributions between tumor types is determined by both mutation and natural selection.

Different tumor types vary greatly in their distribution of driver substitutions. Here, we analyzed how mutation and natural selection contribute to differences in the distribution of KRAS driver substitutions between lung, colon and pancreatic adenocarcinomas. We were able to demonstrate that both differences in mutation and differences in selection drive variation in the distribution of KRAS driver substitutions between tumor types. By accounting for the effects of mutation on the distribution of KRAS driver substitutions, we could identify specific KRAS driver substitutions that are more favored by selection in specific tumor types. Such driver substitutions likely improve fitness most when they occur within the context of the tumor type in which they are preferentially favored. Fitting with this, we found that driver substitutions that are more favored by natural selection in a specific type of tumor tend to associate with worse clinical outcomes specifically in that type of tumor.

Subclonality for BRAF Mutation in Papillary Thyroid Carcinoma Is Associated With Earlier Disease Stage.

CONTEXT: The presence of driver mutations only in a subset of tumor cells within a single lesion, defined as subclonality, is being appreciated as a clinically significant factor. BRAF mutation is the most common driver mutation in papillary thyroid carcinoma (PTC). There are conflicting data in the literature regarding the presence of BRAF mutation subclonality in PTC and its clinical significance. OBJECTIVE: The purpose of the present study was to use a molecular and morphometric approach to determine BRAF clonality status and its clinical-pathological correlates. DESIGN: Fifty-nine cases of PTC were studied. DNA extracted from the tumors underwent deep sequencing to determine the percentage of BRAF mutant allele copies. Additionally, we used computerized morphometry to determine the fraction of tumor cells in each sample. Using both variables, we were able to determine the presence or absence of subclonality for BRAF mutation, which was further correlated with clinical, pathological, and prognostic data. RESULTS: BRAF mutation was found in 49 (83%) cases. The average percentage of tumor cells and of BRAF mutant alleles in the samples were 68.1 ± 9.8 and 26 ± 6.7, respectively. Based on the molecular and morphometric analysis, 11 (24%) cases were found to be subclonal for BRAF mutation. Tumors with subclonal BRAF mutations were significantly smaller compared to tumors with clonal mutation (0.82 ± 0.38 cm vs 1.37 ± 0.57 cm, P = .005) and were less likely to have lymph node metastasis (0% vs 32%, P = .03). CONCLUSIONS: In PTC, subclonality for BRAF mutation is associated with earlier stage. Molecular-morphometric analysis of PTC can provide clonality information with potential clinical significance.

Intratumor Heterogeneity of KRAS Mutation Status in Pancreatic Ductal Adenocarcinoma Is Associated With Smaller Lesions.

OBJECTIVES: Recent studies have demonstrated intratumor heterogeneity (ITH) for genetic mutations in various tumors and suggest that ITH might have significant clinical impact. Because KRAS is the most commonly mutated oncogene in pancreatic ductal adenocarcinoma and has an important role in pancreatic carcinogenesis, the purpose of this study was to evaluate ITH for KRAS gene mutation and its clinical significance. METHODS: Deep sequencing of 47 pancreatic ductal adenocarcinoma cases was used to determine the fraction of KRAS mutant alleles. In addition, computerized morphometry was used to calculate the fraction of tumor cells. After analysis of both results, cases were classified as ITH or as having amplification of mutant KRAS. The presence of ITH was correlated with clinical and pathological factors. RESULTS: KRAS mutation was found in 38 (81%) cases, of which 12 (32%) showed ITH and 9 (23%) were found to have KRAS mutant allele amplification. The presence of ITH was associated with smaller tumors, whereas amplification was associated with higher tumor diameter. CONCLUSIONS: In pancreatic ductal adenocarcinoma, ITH for KRAS gene mutation was associated with smaller tumors. It is possible that, as the tumor progresses, more cells carry this mutation, which leads to more aggressive tumor features.

Human blood-derived endothelial progenitor cells augment vasculogenesis and osteogenesis.

INTRODUCTION: Endothelial progenitor cells (EPC) participate in angiogenesis and osteogenesis, therefore, have the potential to enhance extra-cortical bone formation. AIM: To enhance extra-cortical bone formation following local transplantation of human peripheral blood-derived EPC (hEPC) in a guided bone regeneration (GBR) nude rat calvaria model. MATERIALS AND METHODS: hEPC were isolated from peripheral blood of healthy volunteers. Cells were cultured and characterized by flow cytometry for specific endothelial markers. Following exposure of nude rat calvaria, gold domes were filled with 10(6) hEPC mixed with ßTCP (n = 6). Domes filled with ßTCP served as control (n = 6). Rats were sacrificed after 3 months. New bone formation and blood vessel density were analysed by histology and histomorphometry. Transplanted hEPC were located in the regenerated tissue using immunohistology. RESULTS: Abundant vasculature was observed adjacent to the newly formed bone. According to histomorphometric analysis: blood vessel density was 7.5 folds higher in the hEPC compared with the control group. Similarly, gained extra-cortical bone height (2.46 ± 1.1 mm versus 0.843 ± 0.61 mm, p = 0.01) and bone area fraction (19.42 ± 7.48% versus 4.81 ± 3.93%, p = 0.001) were elevated following hEPC transplantation. Moreover, hEPC expressing human-specific CD31 were integrated into blood vessel walls adjacent to newly formed bone. CONCLUSION: In nude rat GBR calvaria model, transplantation of hEPC significantly enhanced vasculogenesis and osteogenesis.

Co-transplantation of endothelial progenitor cells and mesenchymal stem cells promote neovascularization and bone regeneration.

BACKGROUND: Bone formation relies on sufficient blood supply and osteoprogenitor cells. PURPOSE: The study aims to evaluate the influence of endothelial progenitor cells (EPCs) in combination with mesenchymal stem cells (MSCs) on early vascularization and intramembranous bone regeneration. MATERIALS AND METHODS: Vertical bone regeneration was tested in rat calvarium guided bone regeneration model. Gold domes were filled with a mixture of 5 × 10(5) osteogenic transformed MSC and 5 × 10(5) EPC (EPC/MSC) that were mixed with ß-tricalcium phosphate (ßTCP) scaffold. Domes filled with ßTCP alone served as control. Rats were sacrificed after 4 or 12 weeks. Histomorphometry was used to determine blood vessel (Bv) density, vertical bone height, and bone area in the regenerated tissue. RESULTS: At both time points, new augmented hard tissue filled the space under the dome, and Bv density was higher in the EPC/MSC transplanted group vs control. However, bone height and bone area were similar among the groups 4 weeks posttransplantation, but were doubled in the EPC/MSC transplanted group 12 weeks posttransplantation. CONCLUSIONS: EPC/MSC transplantation increases Bv formation in the early stages of healing that precedes enhancement of extracortical bone regeneration in later stages.

Peripheral blood-derived endothelial progenitor cells enhance vertical bone formation.

BACKGROUND: This study presents a novel cell-based approach for extra-cortical bone regeneration. OBJECTIVE: To enhance vertical bone formation by combining guided bone regeneration and transplantation of peripheral blood-derived endothelial progenitor cells (EPCs) in a rat calvaria model. MATERIALS AND METHODS: EPCs were isolated from peripheral blood of inbred rats. Gold domes (7 mm radius, 5 mm height) were filled with ß-tricalcium phosphate (ßTCP) mixed with 5 × 10(5) EPC. Domes filled with ßTCP served as control (CNT). Rats were sacrificed after 3 months. Vertical bone augmentation was analyzed using histology, histomorphometry, and microcomputed tomography (µCT). RESULTS: In all rats, hard tissue filled the space under the dome. Histomorphometric analysis revealed that EPC transplantations doubled vertical bone height (EPC 4.04 ± 0.22 mm vs CNT 2.29 ± 0.22 mm, p ≤ .001). EPC also caused ∼50% increase in bone area fraction (EPC 47.3 ± 3.1% vs CNT 31.1 ± 2.7%, p ≤ .003). µCT results also showed that bone volume fraction (BV/TV) was higher in EPC group (p = .0169). In both groups, BV/TV declined from the bottom to the top of the samples. No differences in tissue mineral density were found between EPC and CNT groups. CONCLUSION: EPC transplantation significantly improved bone formation especially in the areas that are remote from the original bone.

Adenoma and carcinoma components in colonic tumors show discordance for KRAS mutation.

Activating mutations in KRAS are common events in the pathogenesis of colorectal carcinoma and predict response to treatment with anti-EGFR antibodies. Molecular pathology testing for KRAS mutations has become the standard of practice for patients with metastatic colorectal carcinoma. Despite the known histologic and molecular differences between adenomas and carcinomas, the concordance of KRAS mutation between adenomas and carcinomas has not been established leaving some open questions regarding the appropriate choice of tissue for KRAS mutation analysis and correct interpretation of the test results. To address these questions, we analyzed the concordance of KRAS mutation in 70 tumors that contained both adenoma and carcinoma components (2 cases of intramucosal carcinoma, 66 cases with invasion of the submucosa, and 2 invading the muscularis propria). For each case, DNA was separately isolated from the adenoma and the carcinoma component and analyzed for KRAS mutation using direct sequencing. Overall, 30 (43%) of the adenoma cases and 36 (51%) of the carcinoma cases were positive for KRAS mutation. Of the 70 cases, 16 (23%) showed discordant results. Interestingly, the fraction of discordant cases went down as the depth of carcinoma invasion increased. In summary, we identified significant KRAS mutation discordance between the adenoma and carcinoma component of the lesion. Our results suggest that effort should be made to analyze only the invasive component of the lesion and that caution should be taken when interpreting a result based on DNA extracted from noninvasive elements.

Vertical bone augmentation using different osteoconductive scaffolds combined with barrier domes in the rat calvarium.

PURPOSE: To compare the regenerative potential for vertical bone augmentation of various osteoconductive scaffolds when used in conjunction with barrier domes. MATERIALS AND METHODS: Following exposure and perforation of the calvarium, a gold occlusive dome was filled with the tested scaffold and anchored by fixation screws. Flaps were repositioned and secured. The four treatment groups, three to five rats each, were as follows: Bio-Oss collagen (BOC), ß-tricalcium phosphate (TCP), collagen sponge (COL), and empty domes (C). Rats were sacrificed 8 weeks later, and specimens were prepared for histological and histomorphometric analysis. Vertical bone height and total tissue height were measured. RESULTS: The newly regenerated bone appeared mature, highly vascularized, and with no signs of inflammation. Vertical bone height in the TCP group (mean 2.04 ± 0.2 mm) was greater than all other groups (0.76 ± 0.02, 1.52 ± 0.18, and 1.77 ± 0.61 mm for the BOC, C, and COL, respectively) but significantly only for the BOC group (p = .0145). Total tissue height was significantly higher (p < .0001) in both BOC and TCP groups (4.48 ± 0.23 and 5.5 ± 0.24 mm, respectively) compared with COL (3.22 ± 0.11 mm) and C (2.39 ± 0.3 mm) groups. CONCLUSION: TCP in conjunction with barrier dome resulted in greater vertical bone augmentation in the calvarium of rats.

Mesenchymal stem cells and endothelial progenitor cells stimulate bone regeneration and mineral density.

BACKGROUND: Alveolar bone deficiency is a major clinical problem in maxillofacial reconstructive surgery. The available surgical techniques to enhance extracortical bone augmentation are generally unpredictable and not satisfying. The aim of the present study is to quantify extracortical bone augmentation and tissue mineral density (TMD) after cotransplantation of peripheral blood-derived endothelial progenitor cells (EPCs) and bone marrow-derived mesenchymal stem cells (MSCs) by microcomputed tomography (micro-CT). METHODS: Bone regeneration was tested in the guided bone regeneration rat calvaria model. Gold domes filled with beta tricalcium phosphate (ß-TCP; control [CNT]) or ß-TCP mixed with 5 × 10(5) rat EPCs and 5 × 10(5) rat osteogenic transformed MSCs (EPC/otMSCs) were fixed to the exposed calvaria. Rats were sacrificed after 3 months. Bone volume fraction (BV/TV) and TMD were analyzed using micro-CT. In the middle of the dome, a cylindrical region of interest was defined (it represents the area in which implants are placed) and subdivided into bottom, middle, and top to analyze the effect of the distance from the calvaria on bone formation. RESULTS: In the whole cylinder, BV/TV and TMD were higher in the EPC/otMSC group compared with CNT (BV/TV: 22.9% ± 4.4% versus 29.1 ± 2.2%, P = 0.02; TMD: 937.79 ± 18.68 versus 960.78 ± 5.8 mgHA/ccm, P = 0.03; CNT versus EPC/otMSC, respectively). In each of the three subregions, BV/TV was higher in the EPC/otMSC group compared with CNT (top: 20.25% ± 2.4% versus 23.74% ± 1.5%, P = 0.007; middle: 23.2% ± 4.8% versus 28% ± 2.2%, P = 0.05; bottom: 25.3% ± 7.6% versus 35.7% ± 4.9%, P = 0.02; CNT versus EPC/otMSC, respectively). CONCLUSION: Three-dimensional quantification by micro-CT demonstrated that cotransplantation of EPC/otMSCs significantly improved bone formation and mineral density.

Mesenchymal stem cells combined with barrier domes enhance vertical bone formation.

AIM: To enhance vertical bone formation in a rat calvarium following combination of guided bone regeneration (GBR) and transplantation of bone marrow derived mesenchymal stem cells (bmMSC). MATERIALS AND METHODS: Gold domes (7 mm radius, 5 mm height) were filled with 5 × 10(5) bmMSC or osteogenic transformed bmMSC (otMSC) that were isolated from tibia of inbred rats and mixed with ßTCP. Domes filled with ßTCP served as control. Rats were sacrificed after 3 months. New bone formation was analysed by histology and histomorphometry. RESULTS: In all rats hard tissue filled the space under the dome. In the lower part of the specimens the newly formed mature bone was continuous with the original calvaria, whereas the upper (distal) part of the augmented tissue contained residual scaffold surrounded by connective tissue. Histomorphometric analysis revealed that cell transplantation doubled vertical bone height: (bmMSC 4.13 ± 0.5 mm, otMSC 4.14 ± 0.3 mm ßTCP 2.29 ± 0.22 mm, p ≤ 0.001). Bone area fraction (%) was significantly increased following transplantation of otMSC (47.2 ± 2.5%) when compared with bmMSC (37.3 ± 3.35%) and with ßTCP (31.09 ± 2.7%) (p ≤ 0.031 versus bmMSC, p ≤ 0.0004 versus control) CONCLUSION: In a rat calvaria model transplantation of both otMSC and bmMSC, when combined with GBR significantly enhanced bone formation.

Transplanted blood-derived endothelial progenitor cells (EPC) enhance bridging of sheep tibia critical size defects.

The angiogenic events that accompany bone regeneration function as a "limiting factor" and are the primary regulatory mechanisms that direct the healing process. The general aim of this study was to test whether blood-derived progenitor cells that have endothelial characteristics (EPC), when applied to a large segmental defect, would promote bone regeneration. We established a critical-sized gap platform in sheep tibiae. Our model system takes advantage of the physiological wound healing process that occurs during the first two weeks following injury, and results in the gap being filled with scar tissue. EPC were expanded ex-vivo and 2 x 10(7) cells/0.2 ml were implanted into a wedged-shaped canal excavated in the fibrotic scar tissue. Sham treated sheep served as controls. Bone regeneration was followed every two weeks for three months by X-ray radiography. At the end of the experimental period, the regenerating segments were subjected to micro-computed tomographic (microCT) analysis. While minimal bone formation was detected in sham-treated sheep, six out of seven autologous EPC-transplanted sheep showed initial mineralization already by 2 weeks and complete bridging by 8-12 weeks post EPC transplantation. Histology of gaps 12 weeks post sham treatment showed mostly fibrotic scar tissue. On the contrary, EPC transplantation led to formation of dense and massive woven bone all throughout the defect. The results of this preclinical study open new therapeutic opportunities for the treatment of large scale bone injuries.

Role of bone regeneration and turnover modulators in control of fracture.

Although most fractures heal in accordance with a highly regulated and well-known multistep process, 5%-10% of fractures result in delayed union or nonunion, causing morbidity, prolonged hospitalization, and economic cost for the individual and society. Ongoing research has improved our understanding of genes and molecules that are expressed during fracture healing. This knowledge has been translated into preclinical/clinical trials. Unfortunately, the success of most promising agents, and therefore most preclinically/clinically tested factors, is controversial and frequently disappointing. Taking advantage of our knowledge concerning the temporal events contributing to fracture healing, and as a result of our studies, we suggest that the application of several factors in sequence will intervene in different, crucial crossroads, accelerate remodeling, and result in an improved outcome. These factors will encompass stimulated recruitment and proliferation of stem cells to enlarge the progenitor pool, facilitate its differentiation into mature chondrocytes and osteoblasts, and stimulate cartilage resorption and its remodeling into bone by endochondral ossification--a process in which blood vessels, metalloproteinases, and osteoclasts work in concert to remodel immature bone into mature bone. The aim of this article is to highlight events that contribute to and drive the dynamic healing process in order to help clinicians to find successful, novel treatment protocols.

Fracture repair: modulation of fracture-callus and mechanical properties by sequential application of IL-6 following PTH 1-34 or PTH 28-48.

Fracture healing presents a sequence of three major stages: inflammation and granulation tissue formation, callus formation and remodeling. Our working hypothesis was that fracture-repair might be enhanced by stimulating proliferation of chondrocytes and osteoblasts in the early stages of fracture healing followed by sequential acceleration of the remodeling process. In the present study we employed a novel device developed by us implementing a standardized fracture in rat tibiae. We investigated the effect of PTH 28-48 or PTH 1-34 alone or in sequence combination with IL-6 together with its soluble receptor (IL-6sR) on fracture repair. PTH 28-48 or PTH 1-34 was applied locally into the hematoma of fractures on days 4, 5 and 6 and IL-6+ its soluble receptor on days 7, 9, and 11. Post-fracture callus volume as measured 14 days post-fracture was increased significantly only by PTH 1-34 (20%; P<0.01). When one of the PTH fragments and IL-6+IL-6sR were applied sequentially callus volume was increased significantly (33%; P<0.01). X-rays radiography at 5 weeks post-fracture showed enlarged callus volume following treatment by either PTH fragments alone, and complete union following the sequential injection of both PTH fragments and IL-6+IL-6sR, only. Only the combination of one of the PTH fragments with IL-6+IL-6sR, as measured 6 weeks post-fracture by three point bending, changed dramatically the quality of the regenerating bone as presented by a 300% increase in mechanical resistance when PTH 1-34 was combined and 200% when PTH 28-48 was combined relative to vehicle-treated fractured bones. We conclude that the sequential application of IL-6+IL-6sR with both PTH fragments has the potential of enhancing fracture healing in long bones and should be further explored in preclinical and in clinical studies.

Differential effects of sera from normotensive and hypertensive pregnant women on Ca(2+) metabolism in normal vasular smooth muscle cells.

The clinical features of preeclampsia have been traditionally ascribed to a generalized vascular endothelial cell dysfunction. The present study investigates the effect of sera from preeclamptic women and normal pregnancy on the metabolism of intracellular Ca(2+) concentration ([Ca(2+)](i)) in normal cultured vascular smooth muscle cells (VSMC). Sera were obtained from normotensive pregnant women (NTP) (n = 17), preeclamptic women (PE) (n = 15), pregnant women with chronic (essential) hypertension (pregnant EHT) (n = 8), non-pregnant women with essential hypertension (non-pregnant EHT) (n = 12), and age-matched non-pregnant normotensive women (NNP) (n = 18). Serum (10%) was applied to both primary cultures of rat aortic smooth muscle cells and to the A-10 vascular muscle cell line. Levels of [Ca(2+)](i) were determined fluorometrically. After a 4-h incubation with serum, basal [Ca(2+)](i) was not significantly altered. However, compared with normal pregnant sera, PE sera markedly reduced hormonally induced Ca(2+) transients. Thus, following acute stimulation of rat VSMC (primary cultures) with 10(-8)M angiotensin II, peak [Ca(2+)](i) responses (% increment over baseline) were 443 +/- 22, 184 +/- 18, 259 +/- 12, 274 +/- 23, and 255 +/- 15% in NTP, PE, pregnant EHT, non-pregnant EHT, and NNP, respectively (P <0.01 PE versus NTP, P <0.05 PE versus NNP and pregnant and non-pregnant EHT). These effects of sera on [Ca(2+)](i) were qualitatively reproduced in platelets obtained from healthy volunteers. Also, depolarization-activated Ca(2+) influx in VSMC was affected by the different sera groups in a manner similar to that seen with hormonally induced [Ca(2+)](i) responses. The altered [Ca(2+)](i) changes by PE sera disappeared 5 wk after delivery. The effect of the different sera groups on hormonally triggered Ca(2+) transients in normal VSMC, as well as the normalization of [Ca(2+)](i) responses after delivery, suggest the presence of a circulating serum factor in PE. Inasmuch as [Ca(2+)](i) is the major determinant of VSMC tone, it is possible that consequent to the attenuation of [Ca(2+)](i) responses, this putative circulating factor counterbalances the intense vasoconstriction in PE.