Rainer J. Egli

Hypoxic expansion promotes the chondrogenic potential of articular chondrocytes.

For cell‐based cartilage repair strategies, an ex vivo expansion phase is required to obtain sufficient numbers of cells needed for therapy. Although recent reports demonstrated the central role of oxygen for the function and differentiation of chondrocytes, a beneficial effect of low oxygen concentrations during the expansion of the cells to further improve their chondrogenic capacity has not been investigated. Therefore, freshly harvested bovine articular chondrocytes were grown in two‐dimensional monolayer cultures at 1.5% and 21% O2 and redifferentiation was subsequently induced in three‐dimensional micromass cultures at 1.5%, 5%, and 21% O2. Cells expanded at 1.5% O2 were characterized by low citrate synthase (aerobic energy metabolism)—and high LDH (anaerobic energy metabolism)—activities, suggesting an anaerobic energy metabolism. Collagen type II mRNA was twofold higher in cells expanded at 1.5% as compared to expansion at 21% O2. Micromass cultures grown at 21% O2 showed up to a twofold increase in the tissue content of glycosaminoglycans when formed with cells expanded at 1.5% instead of 21% O2. However, no differences in the levels of transcripts and in the staining for collagen type II protein were observed in these micromass cultures. Hypoxia (1.5% and 5% O2) applied during micromass cultures gave rise to tissues with low contents of glycosaminoglycans only. In vivo, the chondrocytes are adapted to a hypoxic environment. Taking this into account, by applying 1.5% O2 in the expansion phase in the course of cell‐based cartilage repair strategies, may result in a repair tissue with higher quality by increasing the content of glycosaminoglycans.

Cryopreservation of osteochondral allografts: dimethyl sulfoxide promotes angiogenesis and immune tolerance in mice.

Background: Although transplantation of cryopreserved bone allografts has become a routine procedure in orthopaedic surgery, biological and immunological impairment remains an unsolved problem that causes clinical failures. Experimental and clinical evidence has indicated that bone grafts that are revascularized early remain viable and contribute to union at the recipient site. Unprotected cryopreservation, used in most bone banks to reduce graft antigenicity, is associated with complete loss of graft viability, potentially contributing to graft failure. The differences in the survival of various cell types during cryopreservation with use of dimethyl sulfoxide, particularly the increased sensitivity of leukocytes to fast freezing, has resulted in a new approach to modulate immunogenicity. On the basis of this concept, it was proposed that a reduction in the immune response and enhanced revascularization of osteochondral allografts could be achieved by rapid cryopreservation with dimethyl sulfoxide. To test this hypothesis, angiogenesis and immune tolerance were quantified in a murine model with use of intravital microscopy.Methods: Fresh osteochondral tissue and osteochondral tissue that had been cryopreserved with and without dimethyl sulfoxide was transplanted into dorsal skinfold chambers as isografts and as allografts in presensitized and nonsensitized recipient mice. To quantify angiogenesis, the onset of hemorrhages in the vicinity of the grafts and the revascularization of the grafts were determined by means of intravital fluorescence microscopy. To determine the recipients intravascular immune response to the grafts, the leukocyte-endothelium interaction was assessed on the twelfth day after transplantation.Results: Nine of nine fresh isografts were revascularized at a mean (and standard deviation) of 57 ± 33 hours, eight of nine isografts that had been cryopreserved with dimethyl sulfoxide were revascularized at 98 ± 50 hours, and zero of nine isografts that had been cryopreserved without dimethyl sulfoxide were revascularized. Seven of seven fresh allografts were revascularized at 53 ± 6 hours, and ten of ten allografts that had been cryopreserved with dimethyl sulfoxide were revascularized at 82 ± 29 hours. However, signs of revascularization faded in four of the seven fresh allografts whereas reperfusion was maintained in the majority (seven) of the ten grafts frozen in the presence of dimethyl sulfoxide. Similar to the findings associated with unprotected frozen isografts, zero of ten unprotected frozen allografts were revascularized. None of the allografts that had been transplanted into presensitized recipients were revascularized, regardless of whether they had been implanted fresh (nine grafts) or had been implanted after protected (eight grafts) or unprotected (nine grafts) freezing. Quantification of the leukocyte-endothelium interaction revealed a reduction in the intravascular immune response to frozen allografts (both protected and unprotected) compared with fresh allografts.Conclusion: Osteochondral allografts that had been pretreated by cryopreservation with dimethyl sulfoxide demonstrated improved angiogenesis induction and enhanced immune tolerance compared with unprotected frozen grafts. A selective reduction in donor passenger leukocytes is the proposed mechanism underlying this phenomenon.Clinical Relevance: In the absence of presensitization, cryopreservation with dimethyl sulfoxide appears to reduce the immune response to allografts and to enhance their revascularization; in the presence of presensitization, alternatives to allograft transplantation should be considered since the allografts will be exposed to a deleterious immune response.

Transforming Growth Factor Beta Signaling Is Essential for the Autonomous Formation of Cartilage-Like Tissue by Expanded Chondrocytes

Cartilage is a tissue with limited self-healing potential. Hence, cartilage defects require surgical attention to prevent or postpone the development of osteoarthritis. For cell-based cartilage repair strategies, in particular autologous chondrocyte implantation, articular chondrocytes are isolated from cartilage and expanded in vitro to increase the number of cells required for therapy. During expansion, the cells lose the competence to autonomously form a cartilage-like tissue, that is in the absence of exogenously added chondrogenic growth factors, such as TGF-βs. We hypothesized that signaling elicited by autocrine and/or paracrine TGF-β is essential for the formation of cartilage-like tissue and that alterations within the TGF-β signaling pathway during expansion interfere with this process. Primary bovine articular chondrocytes were harvested and expanded in monolayer culture up to passage six and the formation of cartilage tissue was investigated in high density pellet cultures grown for three weeks. Chondrocytes expanded for up to three passages maintained the potential for autonomous cartilage-like tissue formation. After three passages, however, exogenous TGF-β1 was required to induce the formation of cartilage-like tissue. When TGF-β signaling was blocked by inhibiting the TGF-β receptor 1 kinase, the autonomous formation of cartilage-like tissue was abrogated. At the initiation of pellet culture, chondrocytes from passage three and later showed levels of transcripts coding for TGF-β receptors 1 and 2 and TGF-β2 to be three-, five- and five-fold decreased, respectively, as compared to primary chondrocytes. In conclusion, the autonomous formation of cartilage-like tissue by expanded chondrocytes is dependent on signaling induced by autocrine and/or paracrine TGF-β. We propose that a decrease in the expression of the chondrogenic growth factor TGF-β2 and of the TGF-β receptors in expanded chondrocytes accounts for a decrease in the activity of the TGF-β signaling pathway and hence for the loss of the potential for autonomous cartilage-like tissue formation.

Tissue engineering - nanomaterials in the musculoskeletal system

The musculoskeletal tissues bone, cartilage and ligament/tendon are highly structured nanocomposites consisting of nanofibres embedded in a matrix of different composition. Thus, it was a logical step that during the hype of nano in the last decade, nanotechnology and nanomaterials became a hot topic in the field of musculoskeletal repair. Especially the fact that using nanomaterials would encompass a biomimetic approach, thus copying nature, was promising. However, it became evident that using nanomaterials in the repair of musculoskeletal tissues had a longer history than initially thought and its way was paved with failures, which are important to remember when applying current ideas. This current opinion paper summarises some fundamental aspects of nanomaterials to be used for musculoskeletal application and discusses where this field might move to in the near future.

Chondrocytes within Osteochondral Grafts Are More Resistant than Osteoblasts to Tissue Culture at 37°C

ABSTRACT It is proposed that an ideal osteochondral allograft for cartilage repair consists of a devitalized bone but functional cartilage. The different modes of nutrient supply in vivo for bone (vascular support) and cartilage (diffusion) suggest that a modulation of storage conditions could differentially affect the respective cells, resulting in the proposed allograft. For this purpose, osteochondral tissues from porcine humeral heads were either cultured at 37°C for up to 24 hr or stored at 4°C for 24 hr, the temperature at which osteochondral allografts are routinely stored. Functionality of the cells was assessed by in situ hybridization for transcripts encoding collagen types I and II. At 37°C, a time-dependent significant reduction of the bone surface covered with functional cells was observed with only 5% ± 5% coverage left at 24 hr compared with 41% ± 10% at 0 hr. Similarly, cartilage area containing functional cells was significantly reduced from 84% ± 7% at 0 hr to 70% ± 3% after 24 hr. After 24 hr at 4°C, a significantly reduced amount of functional cells covering bone surfaces was observed (27% ± 5%) but not of cells within the cartilage (79% ± 8%). In the applied experimental setup, bone cells were more affected by tissue culture at 37°C than cartilage cells. Even though chondrocytes appear to be more sensitive to 37°C than to 4°C, the substantially reduced amount of functional bone cells at 37°C warrants further investigation of whether a preincubation of osteochondral allografts at 37°C—prior to regular storage at 4°C—might result in an optimized osteochondral allograft with devitalized bone but viable cartilage.

Physiological cartilage tissue engineering effect of oxygen and biomechanics.

In vitro engineering of cartilaginous tissues has been studied for many years, and tissue-engineered constructs are sought to be used clinically for treating articular cartilage defects. Even though there is a plethora of studies and data available, no breakthroughs have been achieved yet that allow for implanting in vivo cultured articular cartilaginous tissues in patients. A review of contributions to cartilage tissue engineering over the past decades emphasizes that most of the studies were performed under environmental conditions neglecting the physiological situation. This is specifically pronounced in the use of bioreactor systems which neither allow for application of near physiomechanical stimulations nor for controlling a hypoxic environment as it is experienced in synovial joints. It is suspected that the negligence of these important parameters has slowed down progress and prevented major breakthroughs in the field. This review focuses on the main aspects of cartilage tissue engineering with emphasis on the relation and understanding of employing physiological conditions.

Histopathology of Cryopreserved Bone Allo- and Isografts: Pretreatment with Dimethyl Sulfoxide

Partial graft cell survival and enhanced graft revascularization have suggested fast freezing using the cryoprotective substance dimethyl sulfoxide (DMSO) as a promising means to improve the biologic function and immune tolerance of allograft bone. This study determines the presence of osteoblasts (colα1(I) mRNA), osteoclasts (TRAP), and cytotoxic T cells (CTLs; GrA mRNA) within pretreated bone grafts 12 days after transplantation. The grafts were transplanted either as isografts, allografts, or allografts in presensitized recipients. In fresh isografts, serving as control, well-formed blood vessels and the highest numbers of viable osteoblasts and osteoclasts were found. In fresh allografts, blood vessels were observed within the marrow cavity and the bone was partially covered by osteoblasts and osteoclasts accompanied by CTLs. In DMSO-pretreated frozen allografts, blood vessels together with osteoblasts were observed in three of five, but in none of five grafts frozen without DMSO. However, infiltration with CTLs was higher in DMSO-pretreated frozen allografts when compared to grafts frozen without DMSO. In presensitized allograft recipients, independent of the pretreatment, in none of the grafts were either blood vessels or osteoblasts found. Thus, fast cryopreservation of bone using DMSO improves vascularization and expression of colα1(I) mRNA (osteoblasts) after allografting when compared to cryopreservation alone, potentially improving graft incorporation. As these grafts were still invaded by CTLs, the long-term effect of DMSO pretreatment needs to be defined.

Differential response of porcine osteoblasts and chondrocytes in cell or tissue culture after 5-aminolevulinic acid-based photodynamic therapy

OBJECTIVE Outcome in osteochondral allografting is limited by the immunological incompatibility of the grafted tissue. Based on a resistance of chondrocytes to photodynamic therapy in cell culture it is proposed that 5-aminolevulinic acid-based photodynamic therapy (5-ALA-PDT) might be used to inactivate bone while maintaining viability of chondrocytes and thus immunomodulate bone selectively. METHODS Chondrocytes and osteoblasts from porcine humeral heads were either isolated (cell culture) or treated in situ (tissue culture). To quantify cytotoxic effects of 5-ALA-PDT (0-20 J/cm(2), 100 mW/cm(2)) an (3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide) (MTT)-assay was used in cell culture and in situ hybridization in tissue culture to assess metabolic active cells (functional osteoblasts: col alpha(1)(I) mRNA, functional chondrocytes: col alpha(1)(II) mRNA). RESULTS In cell culture, survival after 5-ALA-PDT was significantly higher for chondrocytes (5 J/cm(2): 87+/-12% compared to untreated cells) than for osteoblasts (5J/cm(2): 12+/-11%). In tissue culture, the percentage of functional chondrocytes in cartilage showed a decrease after 5-ALA-PDT (direct fixation: 92+/-2%, 20 J/cm(2): 35+/-15%; P<0.0001). A significant decrease in the percentage of bone surfaces covered by functional osteoblasts was observed in freshly harvested (31+/-3%) compared to untreated tissues maintained in culture (11+/-4%, P<0.0001), with no further decrease after 5-ALA-PDT. CONCLUSION Chondrocytes were more resistant to 5-ALA-PDT than osteoblasts in cell culture, while in tissue culture a loss of functional chondrocytes was observed after 5-ALA-PDT. Since osteoblasts - but not chondrocytes - were sensitive to the tissue culture conditions, devitalized bone with functional cartilage might already be achieved by applying specific tissue culture conditions even without 5-ALA-PDT.

In vitro resistance of articular chondrocytes to 5-Aminolevulinic acid based photodynamic therapy.

5‐Aminolevulinic acid based photodynamic therapy (5‐ALA‐PDT) has revealed promising results in the treatment of inflammatory joint diseases due to the sensitivity of inflamed synovial tissue. For 5‐ALA‐PDT to be safe and beneficial for intra‐articular applications, resistance of chondrocytes is essential to prevent cartilage damage. As no data yet exist, the aim of the present study was to assess in vitro the response of the chondrocytes to 5‐ALA‐PDT and to compare with osteoblasts and synovial tissue derived cells.

467 A PHYSIOLOGIC ROBOT REACTOR SYSTEM TO SIMULATE IN VIVO CONDITIONS

Purpose: Success in cartilage tissue engineering and cartilage repair strategies depends on the formation of a hyaline cartilage tissue. In any circumstances, the interaction of at least the four components cells, scaffold/ matrix, biochemical, and biomechanical factors, is of utmost importance to induce or maintain the cells in a chondrocytic phenotype, which is a prerequisite to form hyaline cartilage tissue. Any significant in vitro evaluation, such as testing different cell-scaffold constructs, has to be performed under the harsh conditions encountered in vivo within synovial joints. Therefore, many different bioreactor systems have been developed with the aim to simulate these conditions. However, two main shortcomings have been identified in these systems: (i) the mechanical stimulation units do not operate within a physiological stress range and are limited in the applicable motion pattern, and (ii) most systems lack an ambient control and therefore no hypoxic environment is generated as encountered in synovial joints. We have addressed these shortcomings by designing a fully autonomic modular Physiologic Robot Reactor System (PRRS). Methods: We have engineered a reactor system that comprises a mechanical stimulation unit (MSU), an automatic sample changer (ASC), and an environmental control box (ECB) (Figure 1). The MSU is designed with three linear (orthogonal axes) and one rotational degree of freedom (around z-axis; a rotational component around y-axis is pending). The load generated by the MSU is transferred via an exchangeable plunger on a sample tissue placed in a sample holder (Figure 2). Highly accurate force-feedback and motion systems are controlled by ultrafast Field Programmable Gate Array (FPGA) and real-time components which continuously monitor all system parameters. The ASC is designed as a carrousel providing space for 24 sample holders, which allows for individual piloting of the samples with their own stimulation pattern. The ASC and the MSU are integrated in the ECB in which humidity, temperature, gas composition (O2, CO2), and pressure are actively controlled. In addition, an automated media exchange is also implemented in the system, which enables a prolonged uninterrupted cultivation of sample tissues. Results: The complex physiological motion and load pattern of a knee joint were closely simulated by combining sinusoidal and linear motions. Loading forces of up to 500 N in z-axis were achieved, closely matching the physiological forces encountered in the knee. Within the ECB, the climate is accurately controlled and maintained (deviations of less than 0.1% and 0.1°C from given gas concentrations and temperature, respectively) within the range of the detectors.