Behaviour of a New Composite Mesh for the Repair of Full-Thickness Abdominal Wall Defects in a Rabbit Model

Introduction

Composite biomaterials designed for the repair of abdominal wall defects are composed of a mesh component and a laminar barrier in contact with the visceral peritoneum. This study assesses the behaviour of a new composite mesh by comparing it with two latest-generation composites currently used in clinical practice.

Methods

Defects (7x5cm) created in the anterior abdominal wall of New Zealand White rabbits were repaired using a polypropylene mesh and the composites: Physiomesh^TM; Ventralight^TM and a new composite mesh with a three-dimensional macroporous polyester structure and an oxidized collagen/chitosan barrier. Animals were sacrificed on days 14 and 90 postimplant. Specimens were processed to determine host tissue incorporation, gene/protein expression of neo-collagens (RT-PCR/immunofluorescence), macrophage response (RAM-11-immunolabelling) and biomechanical resistance. On postoperative days 7/14, each animal was examined laparoscopically to quantify adhesions between the visceral peritoneum and implant.

Results

The new composite mesh showed the lowest incidence of seroma in the short term. At each time point, the mesh surface covered with adhesions was greater in controls than composites. By day 14, the implants were fully infiltrated by a loose connective tissue that became denser over time. At 90 days, the peritoneal mesh surface was lined with a stable mesothelium. The new composite mesh induced more rapid tissue maturation than Physiomesh^TM, giving rise to a neoformed tissue containing more type I collagen. In Ventralight^TM the macrophage reaction was intense and significantly greater than the other composites at both follow-up times. Tensile strengths were similar for each biomaterial.

Conclusions

All composites showed optimal peritoneal behaviour, inducing good peritoneal regeneration and scarce postoperative adhesion formation. A greater foreign body reaction was observed for Ventralight^TM. All composites induced good collagen deposition accompanied by optimal tensile strength. The three-dimensional macroporous structure of the new composite mesh may promote rapid tissue regeneration within the mesh.     

In Vivo Dynamic Deformation of Articular Cartilage in Intact Joints Loaded by Controlled Muscular Contractions

When synovial joints are loaded, the articular cartilage and the cells residing in it deform. Cartilage deformation has been related to structural tissue damage, and cell deformation has been associated with cell signalling and corresponding anabolic and catabolic responses. Despite the acknowledged importance of cartilage and cell deformation, there are no dynamic data on these measures from joints of live animals using muscular load application. Research in this area has typically been done using confined and unconfined loading configurations and indentation testing. These loading conditions can be well controlled and allow for accurate measurements of cartilage and cell deformations, but they have little to do with the contact mechanics occurring in a joint where non-congruent cartilage surfaces with different material and functional properties are pressed against each other by muscular forces. The aim of this study was to measure in vivo, real time articular cartilage deformations for precisely controlled static and dynamic muscular loading conditions in the knees of mice. Fifty and 80% of the maximal knee extensor muscular force (equivalent to approximately 0.4N and 0.6N) produced average peak articular cartilage strains of 10.5±1.0% and 18.3±1.3% (Mean ± SD), respectively, during 8s contractions. A sequence of 15 repeat, isometric muscular contractions (0.5s on, 3.5s off) of 50% and 80% of maximal muscular force produced cartilage strains of 3.0±1.1% and 9.6±1.5% (Mean ± SD) on the femoral condyles of the mouse knee. Cartilage thickness recovery following mechanical compression was highly viscoelastic and took almost 50s following force removal in the static tests.     

Effect of Transplanting Various Concentrations of a Composite of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells and Hyaluronic Acid Hydrogel on Articular Cartilage Repair in a Rabbit Model

BackgroundMesenchymal stem cells (MSCs) are known to have therapeutic potential for cartilage repair. However, the optimal concentration of MSCs for cartilage repair remains unclear. Therefore, we aimed to explore the feasibility of cartilage repair by human umbilical cord blood-derived MSCs (hUCB-MSCs) and to determine the optimal concentrations of the MSCs in a rabbit model.MethodsOsteochondral defects were created in the trochlear groove of femur in 55 rabbits. Four experimental groups (11 rabbits/group) were treated by transplanting the composite of hUCB-MSCs and HA with various MSCs concentrations (0.1, 0.5, 1.0, and 1.5 x 10⁷ cells/ml). One control group was left untreated. At 4, 8, and 16 weeks post-transplantation, the degree of cartilage repair was evaluated grossly and histologically.FindingsOverall, transplanting hUCB-MSCs and HA hydrogel resulted in cartilage repair tissue with better quality than the control without transplantation (P = 0.015 in 0.1, P = 0.004 in 0.5, P = 0.004 in 1.0, P = 0.132 in 1.5 x 10⁷ cells/ml). Interestingly, high cell concentration of hUCB-MSCs (1.5×10⁷ cells/ml) was inferior to low cell concentrations (0.1, 0.5, and 1.0 x 10⁷ cells/ml) in cartilage repair (P = 0.394,P = 0.041, P = 0.699, respectively). The 0.5 x 10⁷ cells/ml group showed the highest cartilage repair score at 4, 8 and 16 weeks post transplantation, and followed by 0.1x10⁷ cells/ml group or 1.0 x 10⁷ cell/ml group.ConclusionsThe results of this study suggest that transplantation of the composite of hUCB-MSCs and HA is beneficial for cartilage repair. In addition, this study shows that optimal MSC concentration needs to be determined for better cartilage repair.     

A Phase-Field Model for Articular Cartilage Regeneration in Degradable Scaffolds

Degradable scaffolds represent a promising solution for tissue engineering of damaged or degenerated articular cartilage which due to its avascular nature, is characterized by a low self-repair capacity. To estimate the articular cartilage regeneration process employing degradable scaffolds, we propose a mathematical model as the extension of Olson and Haider’s work (Int. J. Pure Appl. Math. 53:333–353, 2009). The simulated tissue engineering procedure consists in (i) the explant of a cylindrical sample, (ii) the removal of the inner core region, and (iii) the filling of the inner region with hydrogels, degradable scaffolds enriched with nutrients, such as oxygen and glucose. The phase-field model simulates the cartilage regeneration process at the scaffold-cartilage interface. It embeds reaction-diffusion equations, which are used to model the nutrient and regenerated extracellular matrix. The equations are solved using an unconditionally stable hybrid numerical scheme. Cartilage repair processes with full-thickness defects, which are controlled by properties of hydrogel materials and cartilage explant culture based on biological interest are observed. The implemented mathematical model shows the capability to simulate cartilage repairing processes, which can be virtually controlled evaluating hydrogel and cartilage material properties including nutrient supply and defected magnitude. In particular, the adopted methodology is able to explain the regeneration time of cartilage within hydrogel environments. With the numerical scheme, the numerical simulations are demonstrated for the potential improvement of hydrogel structures.     

Rabbit as a Novel Animal Model for Hepatitis E Virus Infection and Vaccine Evaluation

Background

The identification of hepatitis E virus (HEV) from rabbits motivated us to assess the possibility of using rabbits as a non-human primate animal model for HEV infection and vaccine evaluation.

Methodology/Principal Findings

First, 75 rabbits were inoculated with seven strains of genotypes 1, 3, 4, and rabbit HEV, to determine the appropriate strain, administrative route and viral dosage. Second, 15 rabbits were randomly divided into three groups and vaccinated with 0 µg (placebo), 10 µg and 20 µg of HEV candidate vaccine, HEV p179, respectively. After three doses of the vaccination, the rabbits were challenged with 3.3×10⁵ genome equivalents of genotype 4 HEV strain H4-NJ703. The strain of genotype 1 HEV was not found to be infectious for rabbits. However, approximately 80% of the animals were infected by two rabbit HEV strains. All rabbits inoculated with a genotype 3 strain were seroconverted but did not show viremia or fecal viral shedding. Although two genotype 4 strains, H4-NJ153 and H4-NJ112, only resulted in part of rabbits infected, another strain of genotype 4, H4-NJ703, had an infection rate of 100% (five out of five) when administrated intravenously. However, only two out of fifteen rabbits showed virus excretion and seroconversion when inoculated orally with H4-NJ703 of three different dosages. In the vaccine evaluation study, rabbits vaccinated with 20 µg of the HEV p179 produced anti-HEV with titers of 1∶10⁴–1∶10⁵ and were completely protected from infection. Rabbits vaccinated with 10 µg produced anti-HEV with titers of 1∶10³–1∶10⁴ and were protected from hepatitis, but two out of the five rabbits showed virus shedding.

Conclusions/Significance

Rabbits may be served as an alternative to the non-human primate models for HEV infection and vaccine evaluation when certain virus strains, appropriate viral dosages, and the intravenous route of inoculation are selected.     

In Vivo Labeling Method Using a Genetic Construct for Nanoscale Resolution Microscopy

We demonstrate beam scanning-stimulated emission depletion microscopy with in vivo labeled cells. A red emitting fluorescent dye is introduced into membrane protein fused to a multifunctional reporter protein (HaloTag, Promega, Madison, WI) in live cells. This approach allows superresolution stimulated emission depletion imaging without the limitations of immunofluorescence-based staining.     

The Mechanical Behaviour of Chondrocytes Predicted with a Micro-structural Model of Articular Cartilage

The integrity of articular cartilage depends on the proper functioning and mechanical stimulation of chondrocytes, the cells that synthesize extracellular matrix and maintain tissue health. The biosynthetic activity of chondrocytes is influenced by genetic factors, environmental influences, extracellular matrix composition, and mechanical factors. The mechanical environment of chondrocytes is believed to be an important determinant for joint health, and chondrocyte deformation in response to mechanical loading is speculated to be an important regulator of metabolic activity. In previous studies of chondrocyte deformation, articular cartilage was described as a biphasic material consisting of a homogeneous, isotropic, linearly elastic solid phase, and an inviscid fluid phase. However, articular cartilage is known to be anisotropic and inhomogeneous across its depth. Therefore, isotropic and homogeneous models cannot make appropriate predictions for tissue and cell stresses and strains. Here, we modelled articular cartilage as a transversely isotropic, inhomogeneous (TI) material in which the anisotropy and inhomogeneity arose naturally from the microstructure of the depth-dependent collagen fibril orientation and volumetric fraction, as well as the chondrocyte shape and volumetric fraction. The purpose of this study was to analyse the deformation behaviour of chondrocytes using the TI model of articular cartilage. In order to evaluate our model against experimental results, we simulated indentation and unconfined compression tests for nominal compressions of 15%. Chondrocyte deformations were analysed as a function of location within the tissue. The TI model predicted a non-uniform behaviour across tissue depth: in indentation testing, cell height decreased by 43% in the superficial zone and between 11 and 29% in the deep zone. In unconfined compression testing, cell height decreased by 32% in the superficial zone, 25% in the middle, and 18% in the deep zones. This predicted non-uniformity is in agreement with experimental studies. The novelty of this study is the use of a cartilage material model accounting for the intrinsic inhomogeneity and anisotropy of cartilage caused by its microstructure.     

An Optical Section-Assisted In Vivo Rabbit Model for Capsular Bend and Posterior Capsule Opacification Investigation

Purpose

To establish an optical section-assisted in vivo rabbit model for capsular bend and posterior capsule opacification (PCO) investigation.

Methods

A total of 10 rabbits underwent phacoemulsification surgery and intraocular lens (IOL) implantation. On the basis of the relationship between the anterior capsule and IOL, the rabbits were divided into complete overlap and incomplete overlap groups, in which six and four rabbits were included, respectively. The capsular bend optical sections were assessed using ultra-long scan depth optical coherence tomography (UL-OCT), and posterior capsule opacification was evaluated with slit lamp on postoperative day 3, 7, 14, and 28. In addition, histopathological section was used to verify the accuracy of capsular bend type captured by OCT in three rabbits.

Results

Based on the special animal model, six capsular bend types were observed, namely, anterior (A), middle (M), posterior (P), detachment (D), funnel (Fun) and furcate adhesion (Fur). On day 3, capsular bend began to form. On 14 days, the capsular bends were comprised of A, M and D types, which were almost maintained until day 28. Histopathological section findings were consistent with optical sectioning results. In the incomplete and complete groups, the earliest PCO within the optical zone were on day 7 and 28, respectively. The incomplete group exhibited higher incidence and faster PCO on day 7 (p = 0.038) and 14 (p = 0.002).

Conclusions

This animal model not only mimics capsular bend evolution and PCO processes but also produces OCT optical section images equivalent to and more repeatable than histopathology, thereby providing a promising method for the further investigations of PCO.     

重组人中期因子midkine对大鼠膝关节软骨部分损伤的修复作用

目的:通过外源注射不同剂量的重组人中期因子midkine(rhMK),研究其对大鼠膝关节软骨部分损伤的修复作用。方法:雄性SD大鼠双侧膝关节建立软骨部分损伤的动物模型,术后24小时分别向关节腔内注射生理盐水或rhMK (20μg/kg、60μg/kg、180μg/kg)。于术后8周将大鼠全部处死,取材进行组织学观察,从而确定最佳注射剂量;在药代动力学研究中,按最佳注射剂量向正常大鼠膝关节腔内注射rhMK,分别于注射后1小时、1天、3天、6天、9天、12天和15天处死大鼠,检测膝关节软骨组织中rhMK的含量。结果:不同剂量的重组蛋白对膝关节软骨部分损伤均有不同程度的修复作用,其中180μg/kg的剂量效果最佳;以180μg/kg的剂量向正常大鼠膝关节腔内注射rhMK后,经过Kinetica5.0药代动力学软件拟合后,计算得rhMK在软骨组织中的消除相半衰期为8.69天。结论:rhMK对大鼠膝关节软骨部分损伤有明显的修复作用,最佳注射剂量为180μg/kg,最佳注射时间间隔为8天。     

Evaluation of a Novel Thermosensitive Heparin-Poloxamer Hydrogel for Improving Vascular Anastomosis Quality and Safety in a Rabbit Model

Despite progress in the design of advanced surgical techniques, stenosis recurs in a large percentage of vascular anastomosis. In this study, a novel heparin-poloxamer (HP) hydrogel was designed and its effects for improving the quality and safety of vascular anastomosis were studied. HP copolymer was synthesized and its structure was confirmed by Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance spectroscopy (¹H-NMR). Hydrogels containing HP were prepared and their important characteristics related to the application in vascular anastomosis including gelation temperature, rheological behaviour and micromorphology were measured. Vascular anastomosis were performed on the right common carotid arteries of rabbits, and the in vivo efficiency and safety of HP hydrogel to achieve vascular anastomosis was verified and compared with Poloxamer 407 hydrogel and the conventional hand-sewn method using Doppler ultrasound, CT angiograms, scanning electron microscopy (SEM) and histological technique. Our results showed that HP copolymer displayed special gel-sol-gel phase transition behavior with increasing temperature from 5 to 60 °C. HP hydrogel prepared from 18 wt% HP solution had a porous sponge-like structure, with gelation temperature at approximately 38 °C and maximum elastic modulus at 10,000 Pa. In animal studies, imaging and histological examination of rabbit common jugular artery confirmed that HP hydrogel group had similar equivalent patency, flow and burst strength as Poloxamer 407 group. Moreover, HP hydrogel was superior to poloxamer 407 hydrogel and hand-sewn method for restoring the functions and epithelial structure of the broken vessel junctions after operation. By combining the advantages of heparin and poloxamer 407, HP hydrogel holds high promise for improving vascular anastomosis quality and safety.     

Effects of In Vitro Low Oxygen Tension Preconditioning of Adipose Stromal Cells on Their In Vivo Chondrogenic Potential: Application in Cartilage Tissue Repair

Purpose

Multipotent stromal cell (MSC)-based regenerative strategy has shown promise for the repair of cartilage, an avascular tissue in which cells experience hypoxia. Hypoxia is known to promote the early chondrogenic differentiation of MSC. The aim of our study was therefore to determine whether low oxygen tension could be used to enhance the regenerative potential of MSC for cartilage repair.

Methods

MSC from rabbit or human adipose stromal cells (ASC) were preconditioned in vitro in control or chondrogenic (ITS and TGF-β) medium and in 21 or 5% O₂. Chondrogenic commitment was monitored by measuring COL2A1 and ACAN expression (real-time PCR). Preconditioned rabbit and human ASC were then incorporated into an Si-HPMC hydrogel and injected (i) into rabbit articular cartilage defects for 18 weeks or (ii) subcutaneously into nude mice for five weeks. The newly formed tissue was qualitatively and quantitatively evaluated by cartilage-specific immunohistological staining and scoring. The phenotype of ASC cultured in a monolayer or within Si-HPMC in control or chondrogenic medium and in 21 or 5% O₂ was finally evaluated using real-time PCR.

Results/Conclusions

5% O₂ increased the in vitro expression of chondrogenic markers in ASC cultured in induction medium. Cells implanted within Si-HPMC hydrogel and preconditioned in chondrogenic medium formed a cartilaginous tissue, regardless of the level of oxygen. In addition, the 3D in vitro culture of ASC within Si-HPMC hydrogel was found to reinforce the pro-chondrogenic effects of the induction medium and 5% O₂. These data together indicate that although 5% O₂ enhances the in vitro chondrogenic differentiation of ASC, it does not enhance their in vivo chondrogenesis. These results also highlight the in vivo chondrogenic potential of ASC and their potential value in cartilage repair.     

The Repair Response to Osteochondral Implant Types in a Rabbit Model

Current treatments for damaged articular cartilage (i.e., shaving the articular surface, perforation or abrasion of the subchondral bone, and resurfacing with periosteal and perichondrial resurfacing) often produce fibrocartilage, or hyaline-appearing repair that is not sustained over time (Henche 1967, Ligament and Articular Cartilage Injuries. Springer-Verlag, New York, NY, pp. 157–164; Insall 1974, Clin. Orthop. 101: 61–67; Mitchell and Shepard 1976, J. Bone Joint Surg. [Am.] 58: 230–233; O’Driscoll et al. 1986, J. Bone Joint Surg. [Am.] 68: 1017–1035; 1989, Trans. Orthop. Res. Soc. 14: 145; Kim et al. 1991, J. Bone Joint Surg. [Am.] 73: 1301–1315). Autologous chondrocyte transplantation, although promising, requires two surgeries, has site-dependent and patient age limitations, and has unknown long-term donor site morbidity (Brittberg et al. 1994, N Engl. J. Med. 331: 889–895; Minas 2003, Orthopedics 26: 945–947; Peterson et al. 2003, J. Bone Joint Surg. Am. 85-A(Suppl. 2): S17–S24). Osteochondral allografts remain a widely used method of articular resurfacing to delay arthritic progression. The present study compared the histological response to four types of osteochondral implants in a rabbit model: autograft, frozen, freeze-dried, and fresh implants. Specimens implanted in the femoral groove were harvested at 6 and 12 weeks. Results showed similar restoration of the joint surface regardless of implant type, with a trend toward better repair at the later timepoint. As has been observed in other studies (Frenkel et al. 1997, J. Bone Joint Surg. 79B: 281–286; Toolan et al. 1998, J. Biomed. Mater. Res. 41: 244–250), each group in this study had at least one specimen in which a healthy-appearing surface on the implant was not well-integrated with host tissues. Although the differences were not statistically significant, freeze-dried implants at both timepoints had the best histological scores. The osteochondral grafts tested successfully restored the gross joint surface and congruity. At 12 weeks, no significant differences were observed between the various allografts and autologous osteochondral grafts.     

Noninvasive Assessment of Gene Transfer and Expression by In Vivo Functional and Morphologic Imaging in a Rabbit Tumor Model

Purpose

To evaluate the importance of morphology in quantifying expression after in vivo gene transfer and to compare gene expression after intra-arterial (IA) and intra-tumoral (IT) delivery of adenovirus expressing a SSTR2-based reporter gene in a large animal tumor model.

Materials and Methods

Tumor directed IA or IT delivery of adenovirus containing a human somatostatin receptor type 2A (Ad-CMV-HA-SSTR2A) gene chimera or control adenovirus (Ad-CMV-GFP) was performed in VX2 tumors growing in both rabbit thighs. Three days later, ¹¹¹In-octreotide was administered intravenously after CT imaging using a clinical scanner. ¹¹¹In-octreotide uptake in tumors was evaluated the following day using a clinical gamma-camera. Gene expression was normalized to tumor weight with and without necrosis. This procedure was repeated on nine additional rabbits to investigate longitudinal gene expression both 5 days and 2 weeks after adenovirus delivery. CT images were used to evaluate tumor morphology and excised tissue samples were analyzed to determine ¹¹¹In-octreotide biodistribution ex vivo.

Results

VX2 tumors infected with Ad-CMV-HA-SSTR2 had greater ¹¹¹In-octreotide uptake than with control virus (P<0.05). Intra-arterial and intra-tumoral routes resulted in similar levels of gene expression. Longitudinally, expression appeared to wane at 2 weeks versus 5 days after delivery. Areas of necrosis did not demonstrate significant uptake ex vivo. Morphology identified areas of necrosis on contrast enhanced CT and upon excluding necrosis, in vivo biodistribution analysis resulted in greater percent injected dose per gram (P<0.01) and corresponded better with ex vivo biodistribution(r = 0.72, P<0.01, Coefficient of the x-variable = .72) at 2 weeks than without excluding necrosis (P<0.01).

Conclusion

Tumor specificity and high transgene expression can be achieved in tumors via both tumor directed intra-arterial and intra-tumoral delivery in a large animal tumor model. Using clinical machines, morphologic imaging contributes to functional imaging for quantifying SSTR2-based reporter expression in vivo.     

Nanomiemgel - A Novel Drug Delivery System for Topical Application - In Vitro and In Vivo Evaluation

AimThe objective of this study was to formulate and evaluate a unique matrix mixture (nanomiemgel) of nanomicelle and nanoemulsion containing aceclofenac and capsaicin using in vitro and in vivo analyses and to compare it to a marketed formulation (Aceproxyvon).MethodsNanomicelles were prepared using Vitamin E TPGS by solvent evaporation method and nanoemulsion was prepared by high-pressure homogenization method. In vitro drug release and human skin permeation studies were performed and analyzed using HPLC. The efficiency of nanomiemgel as a delivery system was investigated using an imiquimod-induced psoriatic like plaque model developed in C57BL/6 mice.ResultsAtomic Force Microscopy images of the samples exhibited a globular morphology with an average diameter of 200, 250 and 220 nm for NMI, NEM and NMG, respectively. Nanomiemgel demonstrated a controlled release drug pattern and induced 2.02 and 1.97-fold more permeation of aceclofenac and capsaicin, respectively than Aceproxyvon through dermatomed human skin. Nanomiemgel also showed 2.94 and 2.09-fold greater Cmax of aceclofenac and capsaicin, respectively than Aceproxyvon in skin microdialysis study in rats. The PASI score, ear thickness and spleen weight of the imiquimod-induced psoriatic-like plaque model were significantly (p<0.05) reduced in NMG treated mice compared to free drug, NEM, NMI & Aceproxyvon.ConclusionUsing a new combination of two different drug delivery systems (NEM+NMI), the absorption of the combined system (NMG) was found to be better than either of the individual drug delivery systems due to the utilization of the maximum possible paths of absorption available for that particular drug.     

兔在体心脏缺血再灌注模型的制作方法的改进

目的建立兔在体心脏缺血再灌注模型的新方法。方法40只新西兰大白兔随机分为缺血再灌注组（25只）,假手术组（15只）。缺血再灌注组采用＂二线二结＂法结扎心脏左前降支30 min,然后恢复心肌灌注3h;假手术组仅将线从左前降支周围心肌中穿过,但并不结扎。实验中连续描记心电图。两组分别于结扎（穿线）前和再灌注（穿线）后1 h从股静脉取血1 mL测定血清肌钙蛋白。实验结束时取心肌行2,3,5-氯化三苯基四氮唑和苏木精-伊红染色。结果缺血再灌注组心电图存在ST-T的动态演变,再灌注1 h后血清肌钙蛋白浓度明显高于术前（0.47±0.35 vs.0.33±0.31,P=0.002）。两种染色方法均证明存在心肌坏死。结论＂二线二结＂法能够既方便又成功地建立兔在体心脏缺血再灌注模型。     

Tannic Acid and Thiocarbohydrazide as Structural Reinforcement Agents in the Preparation of Rabbit Knee Articular Cartilage for the Scanning Electron Microscope

Samples from seven sectors of the rabbit knee articular cartilage were shaved and prepared for the scanning electron microscope using either tannic acid, thiocarbohydrazide or nothing (control). Surface morphology was found to be more typical to a given sector and less so to a specific preparation procedure. Rough areas were recorded from load-bearing sectors, while smooth areas appeared on load-free ones. However, fibrillations were discerned on control load-bearing sectors only, and pits and humps were never detected. Tannic acid and thiocarbohydrazide may have exerted their structural reinforcing effect on the tissue preservation by enhancing the binding of osmium tetroxide to it, possibly along with that of other soluble tissue constituents.     

Matrix-assisted Autologous Chondrocyte Transplantation for Remodeling and Repair of Chondral Defects in a Rabbit Model

Articular cartilage defects are considered a major health problem because articular cartilage has a limited capacity for self-regeneration ¹. Untreated cartilage lesions lead to ongoing pain, negatively affect the quality of life and predispose for osteoarthritis. During the last decades, several surgical techniques have been developed to treat such lesions. However, until now it was not possible to achieve a full repair in terms of covering the defect with hyaline articular cartilage or of providing satisfactory long-term recovery ^2-4. Therefore, articular cartilage injuries remain a prime target for regenerative techniques such as Tissue Engineering. In contrast to other surgical techniques, which often lead to the formation of fibrous or fibrocartilaginous tissue, Tissue Engineering aims at fully restoring the complex structure and properties of the original articular cartilage by using the chondrogenic potential of transplanted cells. Recent developments opened up promising possibilities for regenerative cartilage therapies.The first cell based approach for the treatment of full-thickness cartilage or osteochondral lesions was performed in 1994 by Lars Peterson and Mats Brittberg who pioneered clinical autologous chondrocyte implantation (ACI) ⁵. Today, the technique is clinically well-established for the treatment of large hyaline cartilage defects of the knee, maintaining good clinical results even 10 to 20 years after implantation ⁶. In recent years, the implantation of autologous chondrocytes underwent a rapid progression. The use of an artificial three-dimensional collagen-matrix on which cells are subsequently replanted became more and more popular ^7-9.MACT comprises of two surgical procedures: First, in order to collect chondrocytes, a cartilage biopsy needs to be performed from a non weight-bearing cartilage area of the knee joint. Then, chondrocytes are being extracted, purified and expanded to a sufficient cell number in vitro. Chondrocytes are then seeded onto a three-dimensional matrix and can subsequently be re-implanted. When preparing a tissue-engineered implant, proliferation rate and differentiation capacity are crucial for a successful tissue regeneration ¹⁰. The use of a three-dimensional matrix as a cell carrier is thought to support these cellular characteristics ¹¹.The following protocol will summarize and demonstrate a technique for the isolation of chondrocytes from cartilage biopsies, their proliferation in vitro and their seeding onto a 3D-matrix (Chondro-Gide, Geistlich Biomaterials, Wollhusen, Switzerland). Finally, the implantation of the cell-matrix-constructs into artificially created chondral defects of a rabbit''s knee joint will be described. This technique can be used as an experimental setting for further experiments of cartilage repair.     

Anti-Hepatoma Activity of a Novel Compound Glaucocalyxin H In Vivo and In Vitro

Glaucocalyxin H (GLH) is a new compound isolated from a traditional Chinese medical herb Isodon japonica var. glaucocalyx which has been used for folk medicine. This study was carried out for the first time to investigate the potential role of GLH in anti-hepatoma activity and underlying mechanisms in it. GLH could inhibit the growth of tumor in mice and induce HepG₂ cells to death as assessed by the tumor reduction assay, toxic assay, morphological change, and survival rate assay. Many antitumor drugs originated from plants could inhibit the growth of tumor by inducing cells to apoptosis. The morphological changes of HepG₂ cells treated with different concentrations of GLH under fluorescence and electron microscope and apoptotic rates were detected to verify its effect on apoptosis. As shown in the study, GLH could induce HepG₂ cells to apoptosis in a dose-dependent manner. Bcl₂ and Bax proteins played important roles in apoptosis and the disequilibrium between Bcl₂ and Bax might result in apoptosis. The expression of Bax protein was upregulated and Bcl₂ protein was downregulated in HepG₂ cells treated with GLH assessed by Western blotting, and they were in a dose-dependent manner. Taken together, GLH can inhibit the growth of hepatoma cells in vivo and in vitro by inducing cell apoptosis due to the decreased Bcl₂ and increased Bax proteins suggesting that GLH could be a potential candidate as an anti-hepatoma agent for the therapeutic treatment of hepatoma.

Electronic supplementary material

The online version of this article (doi:10.1208/s12249-014-0227-3) contains supplementary material, which is available to authorized users.KEY WORDS: apoptosis, Glaucocalyxin H, hepatoma, HepG₂ cell     

In Vitro and In Vivo Isolation of a Novel Rearranged Porcine Circovirus Type 2

Here, we present the first report of a novel rearranged porcine circovirus type 2 (PCV2) strain named BIV, isolated from both in vitro and in vivo sources. The complete circular genome of BIV is 896 nucleotides in length. The data will help us to update current knowledge of the replication of PCV2 viruses in cell culture and of their molecular evolution, as well as their diagnosis.     

Intra-Articular Injections of Polyphenols Protect Articular Cartilage from Inflammation-Induced Degradation: Suggesting a Potential Role in Cartilage Therapeutics

Arthritic diseases, such as osteoarthritis and rheumatoid arthritis, inflict an enormous health care burden on society. Osteoarthritis, a degenerative joint disease with high prevalence among older people, and rheumatoid arthritis, an autoimmune inflammatory disease, both lead to irreversible structural and functional damage to articular cartilage. The aim of this study was to investigate the effect of polyphenols such as catechin, quercetin, epigallocatechin gallate, and tannic acid, on crosslinking type II collagen and the roles of these agents in managing in vivo articular cartilage degradation. The thermal, enzymatic, and physical stability of bovine articular cartilage explants following polyphenolic treatment were assessed for efficiency. Epigallocatechin gallate and tannic acid-treated explants showed >12 °C increase over native cartilage in thermal stability, thereby confirming cartilage crosslinking. Polyphenol-treated cartilage also showed a significant reduction in the percentage of collagen degradation and the release of glycosaminoglycans against collagenase digestion, indicating the increase physical integrity and resistance of polyphenol crosslinked cartilage to enzymatic digestion. To examine the in vivo cartilage protective effects, polyphenols were injected intra-articularly before (prophylactic) and after (therapeutic) the induction of collagen-induced arthritis in rats. The hind paw volume and histomorphological scoring was done for cartilage damage. The intra-articular injection of epigallocatechin gallate and tannic acid did not significantly influence the time of onset or the intensity of joint inflammation. However, histomorphological scoring of the articular cartilage showed a significant reduction in cartilage degradation in prophylactic- and therapeutic-groups, indicating that intra-articular injections of polyphenols bind to articular cartilage and making it resistant to degradation despite ongoing inflammation. These studies establish the value of intra-articular injections of polyphenol in stabilization of cartilage collagen against degradation and indicate the unique beneficial role of injectable polyphenols in protecting the cartilage in arthritic conditions.