In vitro and in vivo evaluations of biodegradable implants for hormone replacement therapy: Effect of system design and PK-PD relationship

This investigation evaluated the feasibility of using subdermally implantable devices fabricated by nonconventional 3-dimensional printing technology for controlled delivery of ethinyl estradiol (EE₂). In vitro release kinetics of EE₂ and in vivo pharmacokinetics pharmacodynamics in ovariectomized New Zealand White rabbits were carried out to study 3 implant prototypes: implant I (single-channel EE₂ distribution in polycaprolactone polymer core), implant II (homogeneous EE₂ distribution in polycaprolactone polymer matrix), and implant III (concentration-gradient EE₂ distribution in polycaprolactone and poly(dl-lactide-co-glycolide) (50∶50 matrix). EE₂ was found to be released from all the implants in a nonlinear pattern with an order of implant III>implant II>implant I. The noncompartmental pharmacokinetic analysis of plasma EE₂ profiles in rabbits indicated a significant difference (p>.05) in C_max, t_max, and mean residence time between implant I and implants II and III, but no difference in the area under the plasma concentration time curves calculated by trapezoidal rule (AUC) among the implants. For pharmacodynamic studies, endogenous follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels were observed to be suppressed following implantation of all implants, which demonstrated that a therapeutically effective dose of EE₂ had been delivered. Furthermore, the noncompartmental analysis of plasma FSH and LH profiles in rabbits showed a significant difference (p<.05) in AUC and the mean residence time between implant III and implants I and II. A good in vivo/in vitro relationship was observed between daily amounts of EE₂ released and plasma profiles of EE₂ for all implants. This relationship suggests that plasma profiles of EE₂ could be predicted from in vitro measurement of daily amount of EE₂ released Therefore, performing in vitro drug release studies may aid in the development of an EE₂ implant with the desired in vivo release rate. Published: September 21, 2001     

Enhanced cartilage tissue engineering by sequential exposure of chondrocytes to FGF-2 during 2D expansion and BMP-2 during 3D cultivation

Bovine calf articular chondrocytes, either primary or expanded in monolayers (2D) with or without 5 ng/ml fibroblast growth factor-2 (FGF-2), were cultured on three-dimensional (3D) biodegradable polyglycolic acid (PGA) scaffolds with or without 10 ng/ml bone morphogenetic protein-2 (BMP-2). Chondrocytes expanded without FGF-2 exhibited high intensity immunostaining for smooth muscle alpha-actin (SMA) and collagen type I and induced shrinkage of the PGA scaffold, thus resembling contractile fibroblasts. Chondrocytes expanded in the presence of FGF-2 and cultured 6 weeks on PGA scaffolds yielded engineered cartilage with 3.7-fold higher cell number, 4.2-fold higher wet weight, and 2.8-fold higher wet weight glycosaminoglycan (GAG) fraction than chondrocytes expanded without FGF-2. Chondrocytes expanded with FGF-2 and cultured on PGA scaffolds in the presence of BMP-2 for 6 weeks yielded engineered cartilage with similar cellularity and size, 1.5-fold higher wet weight GAG fraction, and more homogenous GAG distribution than the corresponding engineered cartilage cultured without BMP-2. The presence of BMP-2 during 3D culture had no apparent effect on primary chondrocytes or those expanded without FGF-2. In summary, the presence of FGF-2 during 2D expansion reduced chondrocyte expression of fibroblastic molecules and induced responsiveness to BMP-2 during 3D cultivation on PGA scaffolds.     

A molecular analysis of matrix remodeling and angiogenesis during long bone development

The replacement of cartilage by bone is the net result of genetic programs that control chondrocyte differentiation, matrix degradation, and bone formation. Disruptions in the rate, timing, or duration of chondrocyte proliferation and differentiation result in shortened, misshapen skeletal elements. In the majority of these skeletal disruptions, vascular invasion of the elements is also perturbed. Our hypothesis is that the processes involved in endochondral ossification are synchronized via the vasculature. The purpose of this study was to examine carefully the events of vascular invasion and matrix degradation in the context of chondrocyte differentiation and bone formation. Here, we have produced a ‘molecular map’ of the initial vascularization of the developing skeleton that provides a framework in which to interpret a wide range of fetal skeletal malformations, disruptions, and dysplasias.     

兔左室壁三层心肌细胞的分离及动作电位、钙和钾电流分布的异质性

探讨兔左室壁三层心肌单个细胞的分离方法以及电生理特征,实验以胶原酶按二步消化法分离兔心肌细胞,其中用剃须刀分离左室游离壁内,中,外三层心肌,采用全细胞膜片钳记录AP和离子电流,结果显示：（1）中层细胞上的动作电位时程明显长于内膜下心肌和外膜下心肌,且存在显著的1相切迹和2相驼峰;（2）中层细胞的Ica,L和Lto较内,外膜下的大,IK,s相反,可见三层心肌细胞上多种电流存在显著差异。     

Dlx5 is a positive regulator of chondrocyte differentiation during endochondral ossification

The process of endochondral ossification in which the bones of the limb are formed after generation of cartilage models is dependent on a precisely regulated program of chondrocyte maturation. Here, we show that the homeobox-containing gene Dlx5 is expressed at the onset of chondrocyte maturation during the conversion of immature proliferating chondrocytes into postmitotic hypertrophying chondrocytes, a critical step in the maturation process. Moreover, retroviral misexpression of Dlx5 during differentiation of the skeletal elements of the chick limb in vivo results in the formation of severely shortened skeletal elements that contain excessive numbers of hypertrophying chondrocytes which extend into ectopic regions, including sites normally occupied by immature chondrocytes. The expansion in the extent of hypertrophic maturation detectable histologically is accompanied by expanded and upregulated domains of expression of molecular markers of chondrocyte maturation, particularly type X collagen and osteopontin, and by expansion of mineralized cartilage matrix, which is characteristic of terminal hypertrophic differentiation. Furthermore, Dlx5 misexpression markedly reduces chondrocyte proliferation concomitant with promoting hypertrophic maturation. Taken together, these results indicate that Dlx5 is a positive regulator of chondrocyte maturation and suggest that it regulates the process at least in part by promoting conversion of immature proliferating chondrocytes into hypertrophying chondrocytes. Retroviral misexpression of Dlx5 also enhances formation of periosteal bone, which is derived from the Dlx5-expressing perichondrium that surrounds the diaphyses of the cartilage models. This suggests that Dlx5 may be involved in regulating osteoblast differentiation, as well as chondrocyte maturation, during endochondral ossification.     

Four distinct chondrocyte populations in the fetal bovine growth plate: highest expression levels of PTH/PTHrP receptor,Indian hedgehog,and MMP-13 in hypertrophic chondrocytes and their suppression by PTH (1-34) and PTHrP (1-40)

Differentiation and growth of chondrocytes in fetal growth plates of vertebrate long bones and ribs appear to occur in a gradual, continuous manner between the resting zone through the proliferation zone, maturation zone, and upper and lower hypertrophic zones, with a continuous increase in cell size up to 10-fold of the volume of a resting chondrocyte. Here we provide evidence, however, that after centrifugation through a continuous Percoll gradient growth plate chondrocytes separate into four distinct cell populations (B1 to B4) which differ markedly in density, size, and gene expression. These populations collect in the absence of any phase borders in the gradient which might serve as concentration barriers. Fractions B1 and B2 contained the largest cells with the lowest buoyant density and showed the highest expression levels for type X collagen (Col X), but only the B1 population expressed high levels of matrix metalloproteinase-13 (collagenase 3). Cells in fraction B3 were significantly smaller and expressed little Col X, while cells in fraction B4 were of similar size to cells in the resting zone without significant Col X expression. The highest levels of parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor (PTHR-1), and Indian hedgehog (Ihh) expression were also found in the hypertrophic fractions B1 and B2 and not in the prehypertrophic fraction B3, as expected from in situ hybridization data on PTHR-1 expression in fetal rodent or chicken growth plates. Incubation of fractions B1 to B3 with the amino-terminal fragments PTH (1-34) or PTHrP (1-40) suppressed the expression of Col X and PTHR-1 by more than 50% and the expression of Ihh nearly completely. In contrast, the mid-regional PTH fragment PTH (28-48) and PTH (52-84) consistently stimulated the expression of PTHR-1 by 10-20% in fractions B1 to B3. These findings confirm the existence of distinct differentiation stages within chondrocytes of the growth plate and support the hypothesis proposed by Vortkamp et al. (Science 273(1996)613) of a regulatory feedback loop of Ihh and PTH/PTHrP fragments controlling the differentiation of proliferating to prehypertrophic chondrocytes, but extend the ability to respond to PTH/PTHrP hypertrophic chondrocytes.     

First synthesize new viruses then regulate their release? The case of the wild rabbit

European wild rabbits originated in southwestern Europe but have been introduced into many other countries world-wide, becoming serious pests in many instances. As a consequence of rabbits being regarded so differently, applied research for their management often has opposing goals, namely their conservation or their control. Furthermore, modern gene technology has led to the concept of using genetically modified myxoma viruses for rabbit management, again with quite contrary aims in mind. In this paper we explain the possible ecological and economic consequences of using these genetically modified viruses inappropriately and we consider whether national and international regulations are sufficient to prevent improper use. If international regulations are inadequate, molecular biologists and ecologists must consider the consequences of their research and advice beyond their own country to avoid unwanted impacts.     

Restriction fragment alleles of the rabbit IGHG genes with reference to the rabbit IGHGCH2 or e locus polymorphism

Among domesticated mammals, rabbit (Oryctolagus cuniculus) is the only species possessing not more than one subclass of immunoglobulin (IgG) antibodies. The rabbit IGHGCH2 or e locus presents two serologically defined alleles, the e14 and e15 allotypes, which are correlated with amino acid variation at the IgG CH2-CH3 interface. Genetic studies, while revealing the adaptive value of this polymorphism, have relied so far entirely upon allo-antisera. Here we show how these alleles can be distinguished by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods. The proposed PCR-RFLP approach allows the monitoring of IGHG locus diversity in rabbit.     

Characterization of subpopulated articular chondrocytes separated by Percoll density gradient

The aim of this study was to develop a method for fractionation of articular chondrocytes from the entire thickness of the tissue. Isolated chondrocytes from rabbit articular cartilage fractionated by centrifugation in a discontinuous Percoll gradient resulted in four cell fractions with two differing properties. The lowest-density fraction consisted mainly of large cells with small nuclei proliferated actively, maintained the chondrocytic phenotype, and secreted larger amounts of proteoglycan. In contrast, the highest-density fraction consisted of small cells with large nuclei proliferated slowly, did not express the chondrocytic phenotype, and produced larger amounts of interleukin 1-induced nitric oxide. Comparing our results with other previous reports, we find that fraction 1 cells are likely originated from the deep layer of the articular cartilage, whereas fraction 4 cells are tentatively categorized as chondrocytes from the superficial layer of cartilage. Centrifugal fractionation of articular chondrocytes via Percoll density gradient permits clear separation of these heterogeneous cells into different phenotypic populations and allows distinguishing of cells from the different layers of articular cartilage. This simple novel method will provide ready separation of articular chondrocytes for the investigation of the pathogenesis of articular cartilage.