W.J.A. Dhert

Effect of local sequential VEGF and BMP-2 delivery on ectopic and orthotopic bone regeneration

Bone regeneration is a coordinated cascade of events regulated by several cytokines and growth factors. Angiogenic growth factors are predominantly expressed during the early phases for re-establishment of the vascularity, whereas osteogenic growth factors are continuously expressed during bone formation and remodeling. Since vascular endothelial growth factor (VEGF) and bone morphogenetic proteins (BMPs) are key regulators of angiogenesis and osteogenesis during bone regeneration, the aim of this study was to investigate if their sequential release could enhance BMP-2-induced bone formation. A composite consisting of poly(lactic-co-glycolic acid) microspheres loaded with BMP-2 embedded in a poly(propylene) scaffold surrounded by a gelatin hydrogel loaded with VEGF was used for the sequential release of the growth factors. Empty composites or composites loaded with VEGF and/or BMP-2 were implanted ectopically and orthotopically in Sprague-Dawley rats (n=9). Following implantation, the local release profiles were determined by measuring the activity of (125)I-labeled growth factors using scintillation probes. After 8 weeks blood vessel and bone formation were analyzed using microangiography, microCT and histology. The scaffolds exhibited a large initial burst release of VEGF within the first 3 days and a sustained release of BMP-2 over the full 56-day implantation period. Although VEGF did not induce bone formation, it did increase the formation of the supportive vascular network (p=0.03) in ectopic implants. In combination with local sustained BMP-2 release, VEGF significantly enhanced ectopic bone formation compared to BMP-2 alone (p=0.008). In the orthotopic defects, no effect of VEGF on vascularisation was found, nor was bone formation higher by the combination of growth factors, compared to BMP-2 alone. This study demonstrates that a sequential angiogenic and osteogenic growth factor release may be beneficial for the enhancement of bone regeneration.

Viable osteogenic cells are obligatory for tissue-engineered ectopic bone formation in goats

In this study we investigated the bone-forming capacity of tissue-engineered (TE) constructs implanted ectopically in goats. As cell survival is questionable in large animal models, we investigated the significance of vitality, and thus whether living cells instead of only the potentially osteoinductive extracellular matrix are required to achieve bone formation. Vital TE constructs of porous hydroxyapatite (HA) covered with differentiated bone marrow stromal cells (BMSCs) within an extracellular matrix (ECM) were compared with identical constructs that were devitalized before implantation. The devitalized implants did contain the potentially osteoinductive ECM. Furthermore, we evaluated HA impregnated with fresh bone marrow and HA only. Two different types of HA granules with a volume of approximately 40 microm were investigated: HA70/800, a microporous HA with 70% interconnected macroporosity and an average pore size of 800 microm, and HA60/400, a smooth HA with 60% interconnected macropores and an average size of 400 microm. Two granules of each type were combined and then treated as a single unit for cell seeding, implantation, and histology. The tissue-engineered samples were obtained by seeding culture-expanded goat BMSCs on the HA and subsequently culturing these constructs for 6 days to allow cell differentiation and ECM formation. To devitalize, TE constructs were frozen in liquid nitrogen according to a validated protocol. Fresh bone marrow impregnation was performed perioperatively (4 mL per implant unit). All study groups were implanted in bilateral paraspinal muscles. Fluorochromes were administered at three time points to monitor bone mineralization. After 12 weeks the units were explanted and analyzed by histology of nondecalcified sections. Bone formation was present in all vital tissue-engineered implants. None of the other groups showed any bone formation. Histomorphometry indicated that microporous HA70/800 yielded more bone than did HA60/400. Within the newly formed bone, the fluorescent labels showed that mineralization had occurred before 5 weeks of implantation and was directed from the HA surface toward the center of the pores. In conclusion, tissue-engineered bone formation in goats can be achieved only with viable constructs of an appropriate scaffold and sufficient BMSCs.

Increased MMP-2 activity during intervertebral disc degeneration is correlated to MMP-14 levels

Intervertebral disc (IVD) degeneration is associated with the increased expression of several matrix metalloproteinases (MMPs), in particular MMP‐2. However, little is known about the actual activity of MMP‐2 in healthy and degenerated discs, or what mechanisms are involved in its activation. A major activation pathway involves complex formation with MMP‐14 and a tissue inhibitor of metalloproteinases‐2 (TIMP‐2). In a series of 56 human IVDs, obtained at autopsy and graded according to the Thompson score (I–V), we analysed whether MMP‐2 activity was increased in different stages of IVD degeneration and to what extent activation was related to the production of MMP‐14 and TIMP‐2. MMP‐2 activation and production were quantified by gelatin zymography. Immunohistochemical staining of MMP‐14 and TIMP‐2 was quantified with a video overlay‐based system. A positive correlation was observed between the amount of active MMP‐2 and pro‐MMP‐2 and degeneration grade (p < 0.001, correlation coefficient (CC) 0.557; and p < 0.001, CC 0.556, respectively). MMP‐2 activity correlated positively with MMP‐14 and less strongly with TIMP‐2 (p = 0.001, CC 0.436; and p = 0.03, CC 0.288, respectively). Moreover, immunopositivity for MMP‐14 correlated to degeneration grade (p = 0.002, CC 0.398). IVD degeneration was associated with the activity of MMP‐2 and the correlation of its activation with MMP‐14 production suggests MMP‐14 activates MMP‐2 during degeneration. As MMP‐14 is capable of activating several other enzymes that are also thought to be involved in IVD degeneration, it may be a key mediator of the degenerative process. Copyright

Application and limitations of chloromethyl-benzamidodialkylcarbocyanine for tracing cells used in bone Tissue engineering.

Bone tissue engineering has the potential to provide us with an autologous bone substitute. Despite extensive research to optimize the technique, little is known about the survival and function of the cells after implantation. To monitor the cells, in vivo labeling is the method of choice. In this study we investigated the use of the fluorescent membrane marker chloromethyl-benzamidodialkylcarbocyanine (CM-Dil) to label cells used in bone tissue engineering. When applying label concentrations up to 50 microM, cells could be labeled efficiently without negative effects on cell vitality, proliferation, or bone-forming capacity. Porous hydroxyapatite scaffolds were seeded with labeled cells, and up to 6 weeks after implantation in nude mice cells could be traced inside tissue-engineered bone. However, contrary to other reports concerning intramembranous labels, transfer of the label from labeled to unlabeled cells was detected. Transfer occurred both in vitro and in vivo between vital cells and between dead and living cells. To determine when in vivo label transfer happened, devitalized, labeled constructs were implanted for various time periods in nude mice. The presence of vital labeled cells inside these constructs, when evaluated at different implantation periods, indicated transfer of the label. Transfer occurred at 7 days postimplantation when 40 microM label was applied, whereas 10 microM labeled constructs showed transfer 10 days after implantation. These findings indicate that CM-Dil label is useful for in vivo tracing of cells for follow-up periods up to 10 days. This makes the label particularly useful for cell survival studies in tissue-engineered implants.

Bone tissue engineering for spine fusion: An experimental study on ectopic and orthotopic implants in rats

Alternatives to the use of autologous bone as a bone graft in spine surgery are needed. The purpose of this study was to examine tissue-engineered bone constructs in comparison with control scaffolds without cells in a posterior spinal implantation model in rats. Syngeneic bone marrow cells were cultured in the presence of bone differentiation factors and seeded on porous hydroxyapatite particles. Seven rats underwent a posterior surgical approach, in which scaffolds with (five rats) or without cells (two rats) were placed on both sides of the lumbar spine. In addition, separate scaffolds were inserted intramuscularly and subcutaneously during the surgical procedure. After 4 weeks, all rats were killed and examined radiographically, by manual palpation of the excised spine and histologically for signs of bone formation or spine fusion. All rats that received cell-seeded scaffolds showed newly formed bone in all three locations, whereas none of the locations in the control rats showed bone formation. The results of this study support the concept of developing tissue-engineering techniques in posterior spine fusion as an alternative to autologous bone.

The Effect of Timing of Mechanical Stimulation on Proliferation and Differentiation of Goat Bone Marrow Stem Cells Cultured on Braided PLGA Scaffolds

Bone marrow stromal cells (BMSCs) have been shown to proliferate and produce matrix when seeded onto braided poly(L-lactide/glycolide) acid (PLGA) scaffolds. Mechanical stimulation may be applied to stimulate tissue formation during ligament tissue engineering. This study describes for the first time the effect of constant load on BMSCs seeded onto a braided PLGA scaffold. The seeded scaffolds were subjected to four different loading regimes: Scaffolds were unloaded, loaded during seeding, immediately after seeding, or 2 days after seeding. During the first 5 days, changing the mechanical environment seemed to inhibit proliferation, because cells on scaffolds loaded immediately after seeding or after a 2-day delay, contained fewer cells than on unloaded scaffolds or scaffolds loaded during seeding (p<0.01 for scaffolds loaded after 2 days). During this period, differentiation increased with the period of load applied. After day 5, differences in cell content and collagen production leveled off. After day 11, cell number decreased, whereas collagen production continued to increase. Cell number and differentiation at day 23 were independent of the timing of the mechanical stimulation applied. In conclusion, static load applied to BMSCs cultured on PLGA scaffolds allows for proliferation and differentiation, with loading during seeding yielding the most rapid response. Future research should be aimed at elucidating the biomechanical and biochemical characteristics of tissue formed by BMSCs on PLGA under mechanical stimulation.

Trends in biomaterials research: An analysis of the scientific programme of the World Biomaterials Congress 2008

The use of artificial biomaterials for the treatment of diseased tissues traces back to more than 2000 years ago, when heavy metals such as gold were extensively used in dentistry [1]. Other early examples of biomaterials include wooden teeth and glass eyes, but generally, this first generation of biomaterials as developed before 1960 had low success rates due to a poor understanding of biocompatibility. A major change came with the ending of World War II, when materials that were originally developed for military purposes became available for general use. Durable, inert metals, ceramics and especially polymers such as PMMA were taken off-the-shelf by surgeons and applied for clinical use. One of the most inspiring examples of this transition from general to specific use of high-performance materials is the development of the first successful total hip replacement by Charnley in 1961, who adopted the use of high-molecular weight polyethylene and PMMA cement from the plastics industry and dentistry, respectively [2]. Generally, however, these industrial materials were not intentionally redesigned for biomedical purposes, and optimal biocompatibility was still considered to be the absence of cytotoxicity [3]. Following these developments, an entirely new field of research was initiated in the 1960s which focused on the design of new biomaterials with improved biological performance. During the second consensus conference on definitions in biomaterials, biomaterials were defined as [4]:

Chondrogenic potential of articular chondrocytes depends on their original location in the knee

OBJECTIVE This study aimed to investigate the regenerative capacity of chondrocytes derived from debrided defect cartilage and healthy cartilage from different regions in the joint to determine the best cell source for regenerative cartilage therapies. METHODS Articular cartilage was obtained from Outerbridge grade III and IV cartilage lesions and from macroscopically healthy weight-bearing and nonweight-bearing (NWB) locations in the knee. Chondrocytes isolated from all locations were either pelleted directly (P0 pellets) or after expansion (P2 pellets) and analyzed for glycosaminoglycan (GAG), DNA, and cartilage-specific gene expression. Harvested cartilage samples and cultured pellets were also analyzed by Safranin O histology and immunohistochemistry for collagen I, II, and X. Immunohistochemical stainings were quantified using a computerized pixel-intensity staining segmentation method. RESULTS After 4 weeks of culture, the P0 pellets derived from grade III or healthy weight-bearing chondrocytes contained more (p<0.015) GAG and GAG normalized per DNA compared to those from grade IV and NWB locations. After expansion, these differences were lost. Cartilage-specific gene expression was higher (p<0.04) in P0 pellets from grade III chondrocytes compared to grade IV chondrocytes. Semiquantitative immunohistochemistry showed a more intense (p<0.033) collagen I and X staining for grade IV debrided cartilage compared to grade III and weight-bearing cartilage. Also, collagen type X staining intensity was higher (p<0.033) in NWB cartilage compared to grade III and weight-bearing regions. CONCLUSION Chondrocytes derived from debrided cartilage perform better than cells from the NWB biopsy site, however, this difference is lost upon expansion. Based thereon, the debrided defect cartilage could be a viable donor site for regenerative cartilage surgery.