Philip Kasten

COMPARISON OF MESENCHYMAL STEM CELLS FROM BONE MARROW AND ADIPOSE TISSUE FOR BONE REGENERATION IN A CRITICAL SIZE DEFECT OF THE SHEEP TIBIA AND THE INFLUENCE OF PLATELET-RICH PLASMA

Aim of the present study was to compare the osteogenic potential of bone marrow derived mesenchymal stem cells (BMSC) and adipose-tissue derived stem cells (ASC) and to evaluate the influence of platelet-rich plasma (PRP) on the osteogenic capacity of ASC in a large animal model. Ovine BMSC (BMSC-group) and ASC (ASC-group) were seeded on mineralized collagen sponges and implanted into a critical size defect of the sheep tibia (n=5 each). In an additional group, platelet-rich plasma (PRP) was used in combination with ASC (PRP-group). Unloaded mineralized collagen (EMPTY-group) served as control (n=5 each). Radiographic evaluation was performed every 2 weeks, after 26 weeks histological analysis was performed. Radiographic evaluation revealed a significantly higher amount of newly formed bone in the BMSC-group compared to the ASC-group at week 10 and compared to EMPTY-group from week 12 (all p<0.05). A superiority on radiographic level concerning bone formation of the PRP-group versus the empty control group was found (p<0.05), but not for the ASC-group. Histological analysis confirmed radiographic evaluation finding analogous significances. In conclusion, ASC seem to be inferior to BMSC in terms of their osteogenic potential but that can partially be compensated by the addition of PRP.

Comparison of human bone marrow stromal cells seeded on calcium-deficient hydroxyapatite, beta-tricalcium phosphate and demineralized bone matrix.

The aim of this study was to compare three resorbable biomaterials regarding seeding efficacy with human bone marrow stromal cells (BMSCs), cell penetration into the matrix, cell proliferation and osteogenic differentiation. Calcium-deficient hydroxyapatite (CDHA), beta-tricalcium phosphate (beta-TCP), and demineralized bone matrix (DBM) were seeded with human BMSCs and kept in human serum and osteogenic supplements for 3 weeks. Morphologic and biochemical evaluations were performed on day 1, 7, 14 and 21. The allograft DBM and CDHA exhibited both an excellent seeding efficacy while the performance of beta-TCP was lower when compared. The total protein content and the values for specific alkaline phosphatase (ALP) increased on all matrices and no significant difference was found for these two markers. BMSCs in monolayer had a significant increase of protein, but not of ALP. Osteocalcin (OC) values increased significantly higher for BMSC in cultures on DBM when compared to CDHA and beta-TCP. The OC levels decreased significantly in the BMSC monolayer culture. BMSCs were found inconsistently within the synthetic materials, whereas in DBM they were found more homogeneously distributed throughout the matrix. All three matrices promoted BMSC proliferation and differentiation to osteogenic cells. DBM allografts seem to be more favorable with respect to cell ingrowth tested by histology, and osteogenic differentiation ascertained by an increase of OC. CDHA with its high specific surface area showed more favorable properties than beta-TCP regarding reproducibility of the seeding efficacy.

Porosity and pore size of β-tricalcium phosphate scaffold can influence protein production and osteogenic differentiation of human mesenchymal stem cells: An in vitro and in vivo study

The interaction of stem cells and ceramics in bone regeneration is still poorly understood. The aim of this study was to examine the influence of the porosity (25%, 65% and 75%) of beta-tricalcium phosphate (TCP) ceramics on osteogenic differentiation of mesenchymal stem cells (MSC) in vitro and in vivo. For the in vitro portion of the study, TCP scaffolds loaded with MSC were kept in osteogenic induction medium for 21 days. For the in vivo portion of the study, scaffolds loaded with undifferentiated MSC were implanted subcutaneously into SCID mice for 8 weeks and compared with similarly implanted controls that were not loaded with MSC. Measurements of total protein as well as specific alkaline phosphatase (ALP) activity were taken as indicators of growth/matrix production and osteogenic differentiation. An increase in the total protein concentration was noted from day 1 to day 21 on the in vitro TCP 65% and TCP 75% scaffolds (p<0.05) with no such increase noted in the TCP 25% specimens. However, the specific alkaline phosphatase activity increased from day 1 to day 21 in all three in vitro specimens (p<0.02) and reached similar levels in each specimen by day 21. In vivo, ALP activity of cell-loaded TCP 65% ceramics was higher when compared with both the TCP 25% and TCP 75% specimens (p<0.046), and higher in the TCP 75% than TCP 25% specimens (p=0.008). Histology revealed mineralization by human cells in the pores of the TCP ceramic scaffolds with a trend toward greater calcification in TCP 65% and 75%. In summary, a higher porosity of TCP scaffolds does not necessarily mean a higher ALP activity in vivo. The distribution and size of the pores, as well as the surface structure, might play an important role for osteogenic differentiation in vivo.

Culture media for the differentiation of mesenchymal stromal cells

Mesenchymal stromal cells (MSCs) can be isolated from various tissues such as bone marrow aspirates, fat or umbilical cord blood. These cells have the ability to proliferate in vitro and differentiate into a series of mesoderm-type lineages, including osteoblasts, chondrocytes, adipocytes, myocytes and vascular cells. Due to this ability, MSCs provide an appealing source of progenitor cells which may be used in the field of tissue regeneration for both research and clinical purposes. The key factors for successful MSC proliferation and differentiation in vitro are the culture conditions. Hence, we here summarize the culture media and their compositions currently available for the differentiation of MSCs towards osteogenic, chondrogenic, adipogenic, endothelial and vascular smooth muscle phenotypes. However, optimal combination of growth factors, cytokines and serum supplements and their concentration within the media is essential for the in vitro culture and differentiation of MSCs and thereby for their application in advanced tissue engineering.

The effect of platelet-rich plasma on healing in critical-size long-bone defects

The role of platelet-rich plasma (PRP) as a promoter of bone healing remains controversial. The hypothesis investigated was that PRP improves bone healing of a critical-size diaphyseal radius defect in a rabbit model. The bone defect was filled with a high-surface ceramic scaffold, calcium-deficient hydroxyapatite (CDHA), with the addition of allogenic PRP, mesenchymal stem cells (MSC) or both. PRP yielded better bone formation than the empty CDHA scaffold as determined by both histology and micro-computer tomography (p<0.05) after 16 weeks, whereas no difference was observed on biomechanical testing. Similar behavior was found in samples with MSC; however, the combination of MSC and PRP did not further improve bone healing. Furthermore, the resorption of CDHA was improved by the addition of PRP, MSC and MSC/PRP, but there were no differences between the groups. The areas of bone formation were greater in areas adjacent to the bone resection areas and towards the intact ulna. In conclusion, PRP improves bone healing in a diaphyseal rabbit model on CDHA and the combination of CDHA. This study supports the allogenic use of PRP for bone healing as an off-the-shelf therapy.

Platelet-rich plasma improves expansion of human mesenchymal stem cells and retains differentiation capacity and in vivo bone formation in calcium phosphate ceramics

Introduction: Mesenchymal stem cells (MSC) applied to bone substitution materials can improve bone healing. Bone formation in biocomposites is highly dependent on the kind of biomaterial, its pre-treatment and the applied cells. Potentially immunogenic or infectious supplements such as fetal calf serum (FCS) should be avoided in cell expansion media. Therefore, we developed an expansion protocol free of xenogenic supplements. Cells expanded with two different media were tested on distinct biomaterials for their bone formation capacity after ectopic implantation in vivo, as well as for their growth rate and differentiation capacity in vitro. Methods: MSC of six donors were expanded with cell expansion medium containing FCS (2%) or platelet-rich plasma (PRP, 3%). Their growth rate and osteogenic, adipogenic and chondrogenic differentiation capacity were compared in vitro. For the in vivo bone formation assay, expanded cells (2 × 105 or 2 × 106) were seeded on calcium-deficient hydroxyapatite (CDHA; n = 12) and on β-tricalcium phosphate (β-TCP; n = 12) blocks, which had been coated with either fibronectin or human serum. They were then implanted subcutaneously in severe combined immunodeficient mice (SCID), harvested after 8 weeks and analysed by histology. Bone formation was assessed by a semi-quantitative bone score, after toluidine blue and alizarin red staining. Human cells were detected by an in situ hybridisation for human-specific alu sequences. Results: PRP-supplemented expansion medium yielded two-fold higher cell numbers compared to medium with FCS (P = 0.046) after 3 weeks (four passages) and retained a similar capacity to differentiate towards the osteogenic, chondrogenic and adipogenic lineage. In vivo bone formation was equal for cells expanded with PRP and FCS and depended on the specific surface area of the carrier. CDHA (specific surface area (SSA) 48 m2/g) showed a significantly better bone formation in deep layers (P = 0.005) than β-TCP (SSA 0.5 m2/g). Fibronectin-coating of the ceramics was slightly superior to coating with human serum (P = 0.045). Conclusions: The replacement of FCS by PRP eliminated risks connected with the use of xenogeneic supplements. It improved expansion of MSC and retained their differentiation and in vivo bone formation capacity in a setting adaptable to autogenous use.

Total shoulder replacement in young and middle-aged patients with glenohumeral osteoarthritis

Our aim in this prospective study was to evaluate the outcome of total shoulder replacement in the treatment of young and middle-aged active patients with primary glenohumeral osteoarthritis. We reviewed 21 patients (21 shoulders) with a mean age of 55 years (37 to 60). The mean follow-up was seven years (5 to 9). The same anatomical, third-generation, cemented implant had been used in all patients. All the patients were evaluated radiologically and clinically using the Constant and Murley score. No patients required revision. In one a tear of the supraspinatus tendon occurred. Overall, 20 patients (95%) were either very satisfied (n = 18) or satisfied (n = 2) with the outcome. Significant differences (p < 0.0001) were found for all categories of the Constant and Murley score pre- and post-operatively. The mean Constant and Murley score increased from 24.1 points (10 to 45) to 64.5 points (39 to 93), and the relative score from 30.4% (11% to 50%) to 83% (54% to 116%). No clinical or radiological signs of loosening of the implant were seen. For young and middle-aged patients with osteoarthritis, third-generation total shoulder replacement is a viable method of treatment with a low rate of complications and excellent results in the mid-term.

Evaluation of Mineralized Collagen and α-Tricalcium Phosphate as Scaffolds for Tissue Engineering of Bone Using Human Mesenchymal Stem Cells

Owing to their plasticity and high proliferation capacity in vitro, mesenchymal stem cells (MSC) isolated from human bone marrow are promising candidates for use in tissue engineering approaches for the repair or replacement of mesenchymal tissues such as bone, cartilage or tendon. In keeping with the tissue engineering concept, these cells are cultivated on three-dimensional (3D) scaffolds to replace 3D tissue defects. Among the scaffolds tested for tissue engineering of bone, those containing phosphorus and calcium, as natural bone does, are the most promising candidates for this purpose. In this study, MSC from five patients were isolated from bone marrow. After in vitro expansion, cells were cultivated and differentiated towards the osteogenic lineage on mineralized collagen sponges and α-tricalcium phosphate (α-TCP). To analyze how appropriate these scaffolds would be for tissue engineering purposes, we established an in vitro characterization system to describe seeding efficiency, cell distribution and proliferation behavior on each scaffold. Real-time reverse transcriptase polymerase chain reaction quantification of important genes involved in osteogenic differentiation [e.g. bone sialoprotein (BSP), bone morphogenic protein 2, alkaline phosphatase and osteocalcin] was used to monitor the differentiation process of cells seeded on mineralized collagen and α-TCP. Using this in vitro characterization, we were able to demonstrate effective 3D growth of MSC on both scaffolds investigated. Improved osteogenic differentiation was observed on the scaffolds as compared to control monolayers. Of the two matrices, mineralized collagen was superior to α-TCP with regard to seeding efficacy (98 vs. 67%, p = 0.0003), increase in osteogenic marker genes (BSP expression on day 24, pcollagen vs. TCP = 0.046) and 3D cell alignment (cell infiltration up to 500 vs. 200 µm). In conclusion, our data suggest that mineralized collagen is a promising candidate for use as a scaffold in tissue engineering of bone.

Influence of Platelet-Rich Plasma on Osteogenic Differentiation of Mesenchymal Stem Cells and Ectopic Bone Formation in Calcium Phosphate Ceramics

Platelet-rich plasma (PRP) contains a mixture of growth factors that play an important role in wound and fracture healing. While PRP enhanced bone formation by autogenous cancellous bone grafts, its influence in combination with different bone substitutes remained unknown. This study evaluated the effect of PRP on osteogenic differentiation and ectopic bone formation of human mesenchymal stem cells (MSC) in distinct resorbable calcium phosphate ceramics. Calcium-deficient hydroxyapatite (CDHA) blocks with a large specific surface area (48 m2/g) and β-tricalcium phosphate (β-TCP) with a low specific surface area (<0.5 m2/g) were loaded with 2 × 105 bone marrow-derived MSC. Half of the specimens were treated with 5-fold concentrated PRP. Biocomposites were implanted subcutaneously into SCID mice or kept under osteogenic culture conditions for 2 weeks before implantation. The addition of PRP increased the specific alkaline phosphatase (ALP) activity (p = 0.012) in undifferentiated MSC/CDHA composites but not in MSC/β-TCP composites. Osteogenic preinduction was ineffective for CDHA and reduced ALP activity of β-TCP composites significantly at explantation. Ectopic bone formation was stronger in MSC/CDHA (7/32) compared to MSC/β-TCP (2/30) composites, but no influence of PRP was evident. In conclusion, the effect of PRP depended on the type of ceramic and the differentiation status of the MSC, and enhanced ALP activity of MSC on the high surface scaffold CDHA only, but PRP did not improve osteogenesis in our setting.

Mid-term survivorship analysis of a shoulder replacement with a keeled glenoid and a modern cementing technique

We have investigated the mid-term outcome of total shoulder replacement using a keeled cemented glenoid component and a modern cementing technique with regard to the causes of failure and loosening of the components. Between 1997 and 2003 we performed 96 total shoulder replacements on 88 patients, 24 men and 64 women with a mean age of 69.7 years (31 to 82). The minimum follow-up was five years and at the time of review 87 shoulders (77 patients) were examined at a mean follow-up of 89.1 months (60 to 127). Cumulative survival curves were generated with re-operations (accomplished and planned), survivorship of the proshesis, loosening of the glenoid (defined as tilt > 5 degrees or subsidence > 5 mm), the presence of radiolucent lines and a Constant score of < 30 as the endpoints. There were two re-operations not involving revision of the implants and the survival rate of the prosthesis was 100.0% for the follow-up period, with an absolute Constant score of > 30 as the endpoint the survival rate was 98%. Radiological glenoid loosening was 9% after five years, and 33% after nine years. There was an incidence of 8% of radiolucent lines in more than three of six zones in the immediate post-operative period, of 37.0% after the first year which increased to 87.0% after nine years. There was no correlation between the score of Boileau and the total Constant score at the latest follow-up, but there was correlation between glenoid loosening and pain (p = 0.001). We found that total shoulder replacement had an excellent mid-term survivorship and clinical outcome. The surgical and cementing techniques were related to the decrease in radiolucent lines around the glenoid compared with earlier studies. One concern, however, was the fact that radiolucent lines increased over time and there was a rate of glenoid loosening of 9% after five years and 33% after nine years. This suggests that the design of the glenoid component, and the implantation and cementing techniques may need further improvement.