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 mu g 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 mu g/d but not for G-CSF 10 mu g/d in Collagen III content (p = 0.035) and a higher Collagen I/III ratio compared to the other groups. Biomechanically G-CSF 1 mu g/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

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 mu g 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 mu g/d but not for G-CSF 10 mu g/d in Collagen III content (p = 0.035) and a higher Collagen I/III ratio compared to the other groups. Biomechanically G-CSF 1 mu g/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

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-1β, MMP-1, and MMP-3 via qPCR. CSM-i showed higher expression of IL-1β, 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-1β expression was higher in CSM-i at higher passage and longer membrane-holding time. IL-1β 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

Additional file 1: of A biomechanical, micro-computertomographic and histological analysis of the influence of diclofenac and prednisolone on fracture healing in vivo

μCT diclo, μCt pred, μCT control, biomech diclo, biomech pred, biomech control. Description of data: single values, mean and median. (XLSX 42 kb