Chondrocyte Culture Parameters for Matrix-Assisted Autologous Chondrocyte Implantation Affect Catabolism and Inflammation in a Rabbit Model.

Cartilage defects represent an increasing pathology among active individuals that affects the ability to contribute to sports and daily life. Cell therapy, such as autologous chondrocyte implantation (ACI), is a widespread option to treat larger cartilage defects still lacking standardization of in vitro cell culture parameters. We hypothesize that mRNA expression of cytokines and proteases before and after ACI is influenced by in vitro parameters: cell-passage, cell-density and membrane-holding time. Knee joint articular chondrocytes, harvested from rabbits (n = 60), were cultured/processed under varying conditions: after three different cell-passages (P1, P3, and P5), cells were seeded on 3D collagen matrices (approximately 25 mm³) at three different densities (2 × 105/matrix, 1 × 106/matrix, and 3 × 106/matrix) combined with two different membrane-holding times (5 h and two weeks) prior autologous transplantation. Those combinations resulted in 18 different in vivo experimental groups. Two defects/knee/animal were created in the trochlear groove (defect dimension: ∅ 4 mm × 2 mm). Four identical cell-seeded matrices (CSM) were assembled and grouped in two pairs: One pair giving pre-operative in vitro data (CSM-i), the other pair was implanted in vivo and harvested 12 weeks post-implantation (CSM-e). CSMs were analyzed for TNF-α, IL-1ß, MMP-1, and MMP-3 via qPCR. CSM-i showed higher expression of IL-1ß, MMP-1, and MMP-3 compared to CSM-e. TNF-α expression was higher in CSM-e. Linearity between CSM-i and CSM-e values was found, except for TNF-α. IL-1ß expression was higher in CSM-i at higher passage and longer membrane-holding time. IL-1ß expression decreased with prolonged membrane-holding time in CSM-e. For TNF-α, the reverse was true. Lower cell-passages and lower membrane-holding time resulted in stronger TNF-α expression. Prolonged membrane-holding time resulted in increased MMP levels among CSM-i and CSM-e. Cellular density was of no significant effect. We demonstrated cytokine and MMP expression levels to be directly influenced by in vitro culture settings in ACI. Linearity of expression-patterns between CSM-i and CSM-e may predict ACI regeneration outcome in vivo. Cytokine/protease interaction within the regenerate tissue could be guided via adjusting in vitro culture parameters, of which membrane-holding time resulted the most relevant one.

A biomechanical, micro-computertomographic and histological analysis of the influence of diclofenac and prednisolone on fracture healing in vivo.

BACKGROUND: Non-steroidal anti-inflammatory drugs (NSAIDs) have long been suspected of negatively affecting fracture healing, although numerous disputes still exist and little data are available regarding diclofenac. Glucocorticoids interfere in this process over a similar and even broader mechanism of action. As many previously conducted studies evaluated either morphological changes or biomechanical properties of treated bones, the conjunction of both structural measures is completely missing. Therefore, it was our aim to evaluate the effects of diclofenac and prednisolone on the fracture callus biomechanically, morphologically and by 3-dimensional (3D) microstructural analysis. METHODS: Femura of diclofenac-, prednisolone- or placebo-treated rats were pinned and a closed transverse fracture was generated. After 21 days, biomechanics, micro-CT (µCT) and histology were examined. RESULTS: The diclofenac group showed significantly impaired fracture healing compared with the control group by biomechanics and µCT (e.g. stiffness: 57.31 ± 31.11 N/mm vs. 122.44 ± 81.16 N/mm, p = 0.030; callus volume: 47.05 ± 15.67 mm3 vs. 67.19 ± 14.90 mm3, p = 0.037, trabecular thickness: 0.0937 mm ± 0.003 vs. 0.0983 mm ± 0.003, p = 0.023), as confirmed by histology. Biomechanics of the prednisolone group showed obviously lower absolute values than the control group. These alterations were confirmed in conjunction with µCT and histology. CONCLUSIONS: The inhibiting effects of both substances were not only mediated by absolute parameters (e.g. breaking load, BV), but we have shown, for the first time, that additional changes occurred in the microstructural bony network. Especially in patients at risk for delayed bone healing (arteriosclerosis, diabetes mellitus, smoking), the administration of these drugs should be weighed carefully.

Growth factor release by vesicular phospholipid gels: in-vitro results and application for rotator cuff repair in a rat model.

BACKGROUND: Biological augmentation of rotator cuff repair is of growing interest to improve biomechanical properties and prevent re-tearing. But intraoperative single shot growth factor application appears not sufficient to provide healing support in the physiologic growth factor expression peaks. The purpose of this study was to establish a sustained release of granulocyte-colony stimulating factor (G-CSF) from injectable vesicular phospholipid gels (VPGs) in vitro and to examine biocompatibility and influence on histology and biomechanical behavior of G-CSF loaded VPGs in a chronic supraspinatus tear rat model. METHODS: G-CSF loaded VPGs were produced by dual asymmetric centrifugation. In vitro the integrity, stability and release rate were analyzed. In vivo supraspinatus tendons of 60 rats were detached and after 3 weeks a transosseous refixation with G-CSF loaded VPGs augmentation (n = 15; control, placebo, 1 and 10 µg G-CSF/d) was performed. 6 weeks postoperatively the healing site was analyzed histologically (n = 9; H&E by modified MOVIN score/Collagen I/III) and biomechanically (n = 6). RESULTS: In vitro testing revealed stable proteins after centrifugation and a continuous G-CSF release of up to 4 weeks. Placebo VPGs showed histologically no negative side effects on the healing process. Histologically in vivo testing demonstrated significant advantages for G-CSF 1 µg/d but not for G-CSF 10 µg/d in Collagen III content (p = 0.035) and a higher Collagen I/III ratio compared to the other groups. Biomechanically G-CSF 1 µg/d revealed a significant higher load to failure ratio (p = 0.020) compared to control but no significant differences in stiffness. CONCLUSIONS: By use of VPGs a continuous growth factor release could be obtained in vitro. The in vivo results demonstrate an improvement of immunohistology and biomechanical properties with a low dose G-CSF application via VPG. The VPG itself was well tolerated and had no negative influence on the healing behavior. Due to the favorable properties (highly adhesive, injectable, biocompatible) VPGs are a very interesting option for biologic augmentation. The study may serve as basis for further research in growth factor application models.

Tetracycline-regulated bone morphogenetic protein 2 gene expression in lentivirally transduced primary rabbit chondrocytes for treatment of cartilage defects.

OBJECTIVE: Treatment of cartilage defects is still challenging, primarily because of the poor self-healing capacity of articular cartilage. Gene therapy approaches have gained considerable attention, but, depending on the vector system used, they can lead to either limited or unrestrained gene expression, and therefore regulation of gene expression is necessary. This study was undertaken to construct an efficient tetracycline (Tet)-regulated, lentivirally mediated system for the expression of growth factor bone morphogenetic protein 2 (BMP-2) in primary rabbit chondrocytes that will allow for the induction and termination of growth factor gene expression once cartilage regeneration is complete. METHODS: Chondrogenic ATDC5 cells and primary rabbit chondrocytes were lentivirally transduced with different tetracycline-on (Tet-On)-regulated, self-inactivating vectors for the induction of expression of enhanced green fluorescent protein (eGFP) or BMP-2, using either a 1-vector system or a 2-vector system. RESULTS: Expression of eGFP was induced on ATDC5 cells and chondrocytes. The highest induction rate and highest level of gene expression were reached when the spleen focus-forming virus long terminal repeat promoter was used to drive the reverse transactivator expression, after the addition of doxycycline, in chondrocytes. An up to 20-fold induction of Tet-mediated BMP-2 expression was observed on ATDC5 cells. The extent of induction and expression level of BMP-2 in chondrocytes were similar between the 1-vector system- and 2-vector system-infected cells (mean +/- SD 15.5 +/- 1.1 ng/ml and 14.6 +/- 0.4 ng/ml, respectively). In addition, prolonged induction and switching-off of BMP-2 expression, as well as repeated induction, were demonstrated. Production of proteoglycans, as shown by Alcian blue staining, demonstrated the functionality of the lentivirally expressed BMP-2 under induced conditions. CONCLUSION: The lentivirally mediated Tet-On system is an effective strategy for efficient, repeatedly inducible expression of BMP-2 in primary rabbit chondrocytes. Therefore, use of this system in in vivo experiments may be a promising approach as a treatment strategy for cartilage defects.

Platelet concentrate vs. saline in a rat patellar tendon healing model.

PURPOSE: To evaluate single centrifuge platelet concentrate as additive for improved tendon healing. Platelet-rich plasma has been reported to improve tendon healing. Single centrifuge platelet concentration may increase platelet concentration enough to positively affect tendon healing. A single centrifuge process will lead to a blood product with increased platelet concentrations which, when added to a surgically created tendon injury, will improve tendon healing when compared with a saline control. METHODS: Lewis rats had a surgical transection of the patellar tendon that was subsequently stabilized with a cerclage suture. Prior to skin closure, the tendon was saturated with either a concentrated platelet solution or saline. At 14 days, all animals were killed, and the extensor mechanism was isolated for testing. Biomechanical testing outputs included ultimate tensile load, stiffness, and energy absorbed. RESULTS: Comparisons between the control group and the concentrated platelet group revealed no differences. A subgroup of the concentrated platelet group consisting of specimens in whom the concentration process was most successful showed significantly higher ultimate tensile load (P < 0.05) and energy absorbed to failure (P < 0.05) when compared to the control group. CONCLUSION: When successful, single centrifuge platelet concentration yields a solution that improves tendon healing when compared with a saline control. Single-spin platelet concentration may yield a biologically active additive that may improve tendon healing, but more studies must be undertaken to ensure that adequate platelet concentration is possible.

Rotator cuff changes in a full thickness tear rat model: verification of the optimal time interval until reconstruction for comparison to the healing process of chronic lesions in humans.

BACKGROUND: The aim of the study was to develop a standardized rat model for chronic rotator cuff tears. Therefore, a time point of degenerative changes that shows comparable histological changes to the chronic tendon tears in humans had to be determined. The rat shoulder has already been described as a standardized model for investigation of the healing behavior in acute supraspinatus lesions. Little data exist about the possibility of generating a chronic rotator cuff lesion. METHODS: We performed a complete detachment of the supraspinatus tendon in 45 Sprague-Dawley rats. After an interval of 3, 6 and 9 weeks (15 rats in each group), the macroscopic and histological changes were analyzed. The histological investigation included atrophy and fatty muscle degeneration, tendon degeneration and the grade of inflammatory changes. For evaluation of tendon degeneration, a modified MOVIN-Score was used. The contralateral shoulder provided as control group. RESULTS: Macroscopically the defect showed an increasing coverage with scar tissue over time with a complete closure in 73% after 9 weeks. The 3 week group showed the highest rate of persisting defects (80%). The atrophy of the supraspinatus muscle decreased from initial slight atrophy to a nearly normal muscle status in the 9 week group. Fatty infiltration was found in three animals per group regardless of the time interval after detachment. Tendon degeneration (modified MOVIN-Score) showed no significant difference between 3 and 6 weeks (p = 0.93) whereas after 9 weeks a significant increased degeneration was found (p < 0.01). In the early phase (3 and 6 weeks), inflammatory cells could be detected more frequently. CONCLUSIONS: The results show that a chronic tear of the human rotator cuff can be imitated in the rat model with some exclusion. The rapid self-healing response in the rat and the fatty infiltration of the human muscle are the main differences. However, tendon degeneration, inflammation and muscle atrophy combined with a persisting defect at 3 weeks after detachment are comparable to the chronic tendon tears in humans. This model can serve as a basis for further research in the field of rotator cuff repair for chronic lesions.

Characterization of eight different tetracyclines: advances in fluorescence bone labeling.

Polychrome sequential labeling with fluorochromes is a standard technique for the investigation of bone formation and regeneration processes in vivo. However, for human application, only tetracycline and its derivates are approved as fluorochromes. Therefore, the aim of this study was to determine the fluorescence characteristics of the different tetracycline derivates to assess the feasibility of sequential in vivo bone labeling using distinguishable fluorochromes. Eight different tetracycline derivates were injected subcutaneously into growing rats as a single dose or sequentially in different combinations. After preparation of resin-embedded undecalcified bone sections, the fluorescence properties of the tetracycline derivates in bone were analyzed using conventional fluorescence microscopy, spectral image analysis and confocal laser scanning microscopy. Each tetracycline derivate exhibited a characteristic fluorescence spectrum, but the differences between them were small. Chlortetracycline could be discriminated reliably from all other derivates and could therefore be combined with any other tetracycline derivate for reliably distinguishable double labeling. Tetracycline itself exhibited the brightest fluorescence of all the investigated derivates. Interestingly, in conventional microscopy the same tetracycline derivative can appear in different colours to the human eye, even if spectral analysis confirmed identical emission peaks. In conclusion, the data suggest that fluorescence double labeling of bone is feasible using appropriate tetracycline derivates in combination with spectral imaging modalities.

Efficient and stable gene transfer of growth factors into chondrogenic cells and primary articular chondrocytes using a VSV.G pseudotyped retroviral vector.

Since efficient transfer of foreign genes into primary articular chondrocytes (CC) is difficult, a VSV.G pseudotyped retroviral vector (Bullet) was developed for marker and growth factor gene transfer. Transduction efficiency was analysed by FACS. BMP2 production was determined by specific hBMP2-ELISA. BMP2 effect on cells regarding proteoglycan production was measured by alcian blue staining and dye quantification. Alkaline phosphatase activity was determined by enzymatic reaction with p-nitrophenyl phosphate at OD 405nm and proliferation rate was analysed by MTT-assay. ATDC5 cells (98.3+/-0.6%SD) were transduced to express the reporter gene eGFP. After 52 weeks 94.7+/-0.6%SD of cells were positive. Retroviral transduction efficiency for nlslacZ exceeded 92.3+/-6.1%SD in rabbit CC and expression remained high after 15 weeks (75.7+/-14.2%SD). ATDC5 cells and CC expressed the growth factor gene hBMP2 after retroviral transduction at different time-points. BMP2 led to an increase in proteoglycan and alkaline phosphatase production. Initially, the proliferation rate detected by MTT-assay increased in both the cell types; afterwards the proliferation rate was similar to controls. The described retroviral vector system achieved high initial transduction rates in ATDC5 cells and CC. Gene transfer was very stable over the time period analysed, rendering it a useful tool for future in vitro and in vivo studies on cartilage remodelling.

Does cefuroxime alter fracture healing in vivo? A micro-computertomographic, biomechanical, and histomorphometric evaluation using a rat fracture model.

Cefuroxime is widely used for antibiotic prophylaxis in orthopedic surgery. However, a recent study indicated a dose-dependent reduction in osteoblast function in vitro. Nevertheless, cell culture might not sufficiently imitate the complex process of bone remodeling. As data concerning possible in vivo interactions of cefuroxime on fracture healing are completely missing, we investigated the following hypothesis: Does Cefuroxime impair bone healing in vivo? Therefore, 34 male Wistar rats were randomised to cefuroxime-treated or control groups, a Kirschner wire was inserted into right femora and closed transverse fractures were produced. Twenty-one days later, the structural properties of the fracture callus in the early fracture healing phase were evaluated via a combination of micro-CT (µCT), biomechanics and histology. µCT demonstrated similar values in the cefuroxime and control group (e.g., callus volume: 67.19 ± 14.90 mm3 vs. 55.35 ± 6.74 mm3 , p = 0.12; density: 635.48 ± 14.81 mg HA/cm3 vs. 647.87 ± 13.01 mg HA/cm3 , p = 0.16). Biomechanically, similar values were again determined between the groups, in terms of both maximum load (77.65 ± 41.82 vs. 78.54 ± 20.52, p = 0.95) and stiffness (122.44 ± 81.16 vs. 123.74 ± 60.08, p = 0.97). Histological findings were consistent with the radiographic results. Thus, no relevant differences between the cefuroxime and control groups could be found and the reported negative effects on osteoblasts in vitro were not confirmed in vivo by using standard concentrations of cefuroxime. In conclusion, cefuroxime can reasonably be recommended in a clinical setting as an antibiotic therapy when fracture healing is involved. However, supraphysiological doses were not evaluated, which may be present when cefuroxime is used as an additive to bone cement and released over time. Therefore, future studies should evaluate the in vivo effects of prolonged high cefuroxime doses on implant incorporation. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2282-2291, 2017.

Polychrome labeling of bone with seven different fluorochromes: enhancing fluorochrome discrimination by spectral image analysis.

Bone formation and remodeling in vivo can be assessed by polychrome labeling using calcium-binding fluorescent dyes. The number of fluorochromes, however, limits this technique due to the fact that with increasing number, fluorescent spectra inevitably overlap, which makes discrimination more difficult. In order to enhance discrimination, we performed spectral image analysis. Non-critical size defects of the femur of male Wistar rats served as a model for bone formation. Eight different fluorochromes (calcein blue, xylenol orange, calcein, alizarine complexone, doxycycline, rolitetracycline, hematoporphyrin, and BAPTA) were administered sequentially subcutaneously every third day starting at day 4 after surgery. Following, bone specimen were embedded in methylmethacrylate and analyzed by spectral image acquisition using a Sagnac type interferometer (ASI, Israel). Seven of the eight applied fluorochromes could be resolved using spectral image examination. With BAPTA, we present a new fluorochrome suitable for bone labeling. Due to the superior sensitivity of the spectral image acquisition, the thickness of the bone sections could be reduced so that 5 mum thick sections could be analyzed. Spectral decomposition and subsequent linear unmixing allows depiction of each individual fluorochrome without interference of any other, enabling a reliable and superior morphometric analysis of labeled regions.

Refixation of the supraspinatus tendon in a rat model--influence of continuous growth factor application on tendon structure.

The purpose was to evaluate histological changes of the supraspinatus tendon (SSP) after refixation under continuous growth factor application over 20 days in comparison to the native healing process. In a chronic rat tendon tear model (15 rats/group), a transosseous SSP refixation was performed and growth factors (control, G-CSF, b-FGF, combination) were continuously released into the subacromial space by an osmotic pump. Tendon healing was evaluated histologically by a modified MOVIN-Score, and Collagen I/III content was determined by immunohistology at 6 weeks. A modified MOVIN sum score showed significant lower counts for G-CSF and b-FGF in comparison to the control group (p = 0.050/p = 0.027) and the combined group (p = 0.050/p = 0.043). Collagen III was significantly reduced in the combined group compared to the control group (p = 0.028). Collagen I showed no significant differences. The Collagen I/III ratio was nearly doubled for b-FGF and the combined group compared to the control. At the study endpoint, 33% of pump dislocations were detected. The continuous application of both isolated growth factors (G-CSF/b-FGF) achieved improved tendon-remodeling. However, the continuous application via an osmotic pump showed a relative high dislocation rate when applied in the rat model.

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.

Treatment of osteochondral defects in the rabbit's knee joint by implantation of allogeneic mesenchymal stem cells in fibrin clots.

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular cartilage and subchondral bone in recent years led to increased attention to restoration of the entire osteochondral unit. In comparison to chondral lesions the regeneration of osteochondral defects is much more complex and a far greater surgical and therapeutic challenge. The damaged tissue does not only include the superficial cartilage layer but also the subchondral bone. For deep, osteochondral damage, as it occurs for example with osteochondrosis dissecans, the full thickness of the defect needs to be replaced to restore the joint surface (1). Eligible therapeutic procedures have to consider these two different tissues with their different intrinsic healing potential (2). In the last decades, several surgical treatment options have emerged and have already been clinically established (3-6). Autologous or allogeneic osteochondral transplants consist of articular cartilage and subchondral bone and allow the replacement of the entire osteochondral unit. The defects are filled with cylindrical osteochondral grafts that aim to provide a congruent hyaline cartilage covered surface (3,7,8). Disadvantages are the limited amount of available grafts, donor site morbidity (for autologous transplants) and the incongruence of the surface; thereby the application of this method is especially limited for large defects. New approaches in the field of tissue engineering opened up promising possibilities for regenerative osteochondral therapy. The implantation of autologous chondrocytes marked the first cell based biological approach for the treatment of full-thickness cartilage lesions and is now worldwide established with good clinical results even 10 to 20 years after implantation (9,10). However, to date, this technique is not suitable for the treatment of all types of lesions such as deep defects involving the subchondral bone (11). The sandwich-technique combines bone grafting with current approaches in Tissue Engineering (5,6). This combination seems to be able to overcome the limitations seen in osteochondral grafts alone. After autologous bone grafting to the subchondral defect area, a membrane seeded with autologous chondrocytes is sutured above and facilitates to match the topology of the graft with the injured site. Of course, the previous bone reconstruction needs additional surgical time and often even an additional surgery. Moreover, to date, long-term data is missing (12). Tissue Engineering without additional bone grafting aims to restore the complex structure and properties of native articular cartilage by chondrogenic and osteogenic potential of the transplanted cells. However, again, it is usually only the cartilage tissue that is more or less regenerated. Additional osteochondral damage needs a specific further treatment. In order to achieve a regeneration of the multilayered structure of osteochondral defects, three-dimensional tissue engineered products seeded with autologous/allogeneic cells might provide a good regeneration capacity (11). Beside autologous chondrocytes, mesenchymal stem cells (MSC) seem to be an attractive alternative for the development of a full-thickness cartilage tissue. In numerous preclinical in vitro and in vivo studies, mesenchymal stem cells have displayed excellent tissue regeneration potential (13,14). The important advantage of mesenchymal stem cells especially for the treatment of osteochondral defects is that they have the capacity to differentiate in osteocytes as well as chondrocytes. Therefore, they potentially allow a multilayered regeneration of the defect. In recent years, several scaffolds with osteochondral regenerative potential have therefore been developed and evaluated with promising preliminary results (1,15-18). Furthermore, fibrin glue as a cell carrier became one of the preferred techniques in experimental cartilage repair and has already successfully been used in several animal studies (19-21) and even first human trials (22). The following protocol will demonstrate an experimental technique for isolating mesenchymal stem cells from a rabbit's bone marrow, for subsequent proliferation in cell culture and for preparing a standardized in vitro-model for fibrin-cell-clots. Finally, a technique for the implantation of pre-established fibrin-cell-clots into artificial osteochondral defects of the rabbit's knee joint will be described.

Bisphosphonate related osteonecrosis of the jaw: A minipig large animal model.

Bisphosphonate related osteonecrosis of the jaw (BRONJ) is rare but potentially severe, and the etiopathology and risk factors are poorly defined. To date, it has not been possible to induce BRONJ in a large animal model, a shortfall this study aims to redress. Ten two-year-old adult Göttingen minipigs were split into two groups. Five pigs (group 1) were administered intravenously a weekly dose of a bisphosphonate (zoledonate 0.05mg/kg body weight, approximating the oncologic dose in humans) and five pigs (group 2) served as controls. After 6 weeks, tooth extractions were performed in the upper and lower jaw (both groups) and the bisphosphonate administration continued for a further 10 weeks (group 1 only). Clinical and blood parameters were monitored throughout the entire experiment; thereafter, the jaw-bones were subjected to macroscopic, radiological (CT) and histological investigations. Whilst the extraction sites in the control group healed within two weeks, all animals in the bisphosphonate group exhibited exposed bone and impaired wound healing, indicators that are synonymous of macroscopically advanced osteonecrosis. Radiological and in particular histological investigations confirmed the presence of BRONJ in the animals from group 1. This paper demonstrates that the administration of bisphosphonates, in combination with tooth extractions, induces BRONJ in a minipig model. The ability to study BRONJ in miniature pigs, animals with a bone structure not dissimilar to humans, may improve our knowledgebase regarding the etiopathology, the prophylaxis and potentially uncover new therapies of BRONJ.

The dependence of autologous chondrocyte transplantation on varying cellular passage, yield and culture duration.

Matrix-assisted chondrocyte transplantation (m-ACI) still lacks any standardization in its execution in terms of cell passage (P), cell yield (C) and in vitro membrane-holding time (T). It was the goal of this study to analyze the effect of shifting cell culture parameters (P, C, T) on the in vitro as well as in vivo effort of a regulated animal m-ACI. Autologous rabbit knee articular chondrocytes were seeded within bilayer collagen I/III 3-D matrices in variation of P, C and T. Each time, 2 PCT-identical by 2 PCT-identical cell-matrix-constructs (CMC)/animal were created. Simultaneously 2 (PCT-distinct) were re-implanted (CMC-e) autologous into artificial trochlear pristine chondral defects in vivo to remain for 12 weeks while the remaining 2 were harvested (CMC-i) for immediate in vitro analysis at the time of transplantation of their identical twins. mRNA of both, CMC-e regenerates and CMC-i membranes, was analyzed for Collagen-1,-2,-10, COMP, Aggrecan, Sox9 expression by use of a mixed linear model, multiple regression analysis. Generally, CMC-i values were higher than CMC-e values for differentiation targets; the opposite was true for dedifferentiation targets. Regarding individual gene expression, in vivo regenerate cell-matrix properties were significantly dependent on initial cell-matrix in vitro values as a sign of linearity. The parameter membrane-holding time (T) had strongest effects on the resulting mRNA expression with slightly less impact of the parameter passage (P), whereas cell yield (C) had clearly less effects. Noting differences between in vitro and in vivo data, in general, optimal expression patterns concerning chondrogenic differentiation were achieved by few passages, medium cellular yield, short membrane-holding time. Clinical m-ACI may benefit from optimal orchestration of the cell culture parameters passage, yield and time.

Tissue engineering of the anterior cruciate ligament-sodium dodecyl sulfate-acellularized and revitalized tendons are inferior to native tendons.

The acellularization of tendons using detergents (sodium dodecyl sulfate, Triton-X, tri-nitro-butyl-phosphate) is a new source of scaffolds for tissue engineering in anterior cruciate ligament (ACL) repair. In vitro testing demonstrated that acellular tendon scaffolds are biocompatible and show good biomechanical properties, but in vivo confirmation of these results is not yet available. Therefore, the aim of this study was to see in vivo if an acellular allogenic construct colonized with autologous fibroblasts improves the quality of ACL reconstruction. ACL replacement was performed in 31 New Zealand White rabbits using a standardized model. Fifteen animals received autologous semitendinosus tendon, whereas 16 animals were treated with a tissue-engineered construct. This construct was made by acellularization of allogenic semitendinosus tendons using sodium dodecyl sulfate and subsequent in vitro colonization with autologous fibroblasts. Eight weeks postoperatively, macroscopic, biomechanical (ultimate load to failure, elongation, stiffness; n = 8/9), and histological (n = 5) examinations were performed. Biomechanical testing showed decreasing strength of the constructs at 8 weeks after implantation compared with the direct postsurgical strength. However, tissue-engineered constructs (F = 19.7 +/- 20.3 N) were significantly weaker than autologous tendons (F = 61.2 +/- 31.2 N). Histologically, the autologous tendons showed signs of partial necrosis and tissue remodeling. The tissue-engineered constructs exhibited an inflammatory reaction and showed both repopulated and acellular regions. In conclusion, in vivo results were much more unfavorable than in vitro results had suggested. Further studies have to be performed to test if modifications of the acellularization process yield better results in vivo.

The influence of the stable expression of BMP2 in fibrin clots on the remodelling and repair of osteochondral defects.

Growth factors like BMP2 have been tested for osteochondral repair, but transfer methods used until now were insufficient. Therefore, the aim of this study was to analyse if stable BMP2 expression after retroviral vector (Bullet) transduction is able to regenerate osteochondral defects in rabbits. Fibrin clots colonized by control or BMP2-transduced chondrocytes were generated for in vitro experiments and implantation into standardized corresponding osteochondral defects (n=32) in the rabbit trochlea. After 4 and 12 weeks repair tissue was analysed by histology (HE, alcian-blue, toluidine-blue), immunohistochemistry (Col1, Col2, aggrecan, aggrecan-link protein), ELISA (BMP2), and quantitative RT-PCR (BMP2, Col1, Col2, Col10, Cbfa1, Sox9). In vitro clots were also analysed by BMP2-ELISA, histology (alcian-blue), quantitative RT-PCR and in addition by electron microscopy. BMP2 increased Col2 expression, proteoglycan production and cell size in vitro. BMP2 transduction by Bullet was efficient and gene expression was stable in vivo over at least 12 weeks. Proteoglycan content and ICRS-score of repair tissue were improved by BMP2 after 4 and 12 weeks and Col2 expression after 4 weeks compared to controls. However, in spite of stable BMP2 expression, a complete repair of osteochondral defects could not be demonstrated. Therefore, BMP2 is not suitable to regenerate osteochondral lesions completely.

A fibrin glue composition as carrier for nucleic acid vectors.

PURPOSE: Gene delivery from biomaterials has become an important tool in tissue engineering. The purpose of this study was to generate a gene vector-doted fibrin glue as a versatile injectable implant to be used in gene therapy supported tissue regeneration. METHODS: Copolymer-protected polyethylenimine(PEI)-DNA vectors (COPROGs), naked DNA and PEI-DNA were formulated with the fibrinogen component of the fibrin glue TISSUCOL and lyophilized. Clotting parameters upon rehydration and thrombin addition were measured, vector release from fibrin clots was determined. Structural characterizations were carried out by electron microscopy. Reporter and growth factor gene delivery to primary keratinocytes and chondrocytes in vitro was examined. Finally,chondrocyte colonized clots were tested for their potency in cartilage regeneration in a osteochondral defect model. RESULTS: The optimized glue is based on the fibrinogen component of TISSUCOL, a fibrin glue widely used in the clinics, co-lyophilized with copolymer-protected polyethylenimine(PEI)- DNA vectors (COPROGs). This material, when rehydrated, forms vector-containing clots in situ upon thrombin addition and is suitable to mediate growth factor gene delivery to primary keratinocytes and primary chondrocytes admixed before clotting. Unprotected PEI-DNA in the same setup was comparatively unsuitable for clot formation while naked DNA was ineffective in transfection. Naked DNA was released rapidly from fibrin clots (>70% within the first seven days) in contrast to COPROGs which remained tightly immobilized over extended periods of time (0.29% release per day). Electron microscopy of chondrocytecolonized COPROG-clots revealed avid endocytotic vector uptake. In situ BMP-2 gene transfection and subsequent expression in chondrocytes grown in COPROG clots resulted in the upregulation of alkaline phosphatase expression and increased extracellular matrix formation in vitro. COPROG-fibrinogen preparations with admixed autologous chondrocytes when clotted in situ in osteochondral defects in the patellar grooves of rabbit femura gave rise to luciferase reporter gene expression detectable for two weeks (n=3 animals per group). However, no significant improvement in cartilage formation in osteochondral defects filled with autologous chondrocytes in BMP-2-COPROG clots was achieved in comparison to controls (n=8 animals per group). CONCLUSIONS: COPROGs co-lyophilized with fibrinogen are a simple basis for an injectable fibrin gluebased gene-activated matrix. The preparation can be used is complete analogy to fibrin glue preparations that are used in the clinics. However, further improvements in transgene expression levels and persistence are required to yield cartilage regeneration in the osteochondral defect model chosen in this study.