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Tissue engineering of ligaments based on biomechanically suitable biomaterials combined with autologous cells may provide a solution for the drawbacks associated with conventional graft material. The aim of the present study was to investigate the contribution of recombinant human transforming growth factor beta 1 (rhTGF-beta1) and growth differentiation factor (GDF)-5, known for their role in connective tissue regeneration, to proliferation and matrix production by human bone marrow stromal cells (BMSCs) cultured onto woven, bioabsorbable, 3-dimensional (3D) poly(lactic-co-glycolic acid) scaffolds. Cells were cultured for 12 days in the presence or absence of these growth factors at different concentrations. Human BMSCs attached to the suture material, proliferated, and synthesized extracellular matrix rich in collagen type I and collagen III. No differentiation was demonstrated toward cartilage or bone tissue. The addition of rhTGF-beta1 (1-10 ng/mL) and GDF-5 (10-100 ng/mL) increased cell content (p < 0.05), but only TGF-beta1 also increased total collagen production (p < 0.05) and collagen production per cell, which is a parameter indicating differentiation. In conclusion, stimulation with rhTGF-beta1, and to a lesser extent with GDF-5, can modulate human BMSCs toward collagenous soft tissue when applied to a 3D hybrid construct. The use of growth factors could play an important role in the improvement of ligament tissue engineering.

Various in vivo and in vitro studies suggest that joint homeostasis may have a crucial effect on the quality of regeneration tissue resulting from cartilage tissue engineering techniques. The goal of the current study was to evaluate the effect of synovial fluid (SF) from injured knee joints on in vitro chondrogenesis. Chondrocytes were isolated from a healthy human femoral condyle (post-mortem) and expanded in monolayer for 2 passages. Subsequently, the chondrocytes were redifferentiated for 14 days on collagencoated filters, cultured either in the presence or absence of 10% SF. SF was obtained from 12 injured human knee joints. After 14 days of culture, SF supplementation resulted in a significant downregulation of final proteoglycan (PG) content (7.3 +/- 1.8 mg versus 15.6 +/- 1.3 mg; p = 0.0001), PG content normalized to DNA (0.7 +/- 0.5 mg/microg versus 3.0 +/- 0.6 mg/microg; p < 0.05), relative collagen type II mRNA levels normalized to GAPDH mRNA levels (0.2 +/- 0.3 versus 7.0 +/- 5.6; p < 0.001), and differentiation index (collagen type II/I mRNA ratio; 0.1 +/- 0.2 versus 6.0 +/- 2.9; p < 0.001) as compared to control culture conditions. Additionally, SF-supplemented media resulted in significantly increased cellularity, as reflected by DNA content, compared with control media (1,369 +/- 683 microg versus 514 +/- 72 microg; p < 0.0001). Morphology, and collagen type I, X, and aggrecan mRNA levels were not significantly affected. In conclusion, this study demonstrates that SF from injured human knee joints significantly affects in vitro chondrogenesis and therefore may provide a viable target for future improvement of ACT by refinement of culture techniques, patient selection, or pretreatment of affected joints to restore joint homeostasis.

Cartilage regeneration based on isolated and culture-expanded chondrocytes is studied in a variety of in vitro models, but with varying morphological quality of tissue synthesized. The goal of the present study was to investigate the extent of the influence of expansion and redifferentiation conditions on final tissue morphology by comparing 2 expansion and redifferentiation methods. Chondrocytes from 9 human donors were expanded in medium without growth factor supplementation (basic expansion condition [BEC]) or in medium with basic fibroblast growth factor (bFGF) supplementation (growth factor supplemented expansion condition [GFSEC]). After expansion, cells were either redifferentiated in pellet culture or seeded on collagen type II-coated filters. Post-expansion mRNA levels of collagen type I and II and Sox-5, -6, and 9, measured by semiquantitative real-time polymerase chain reaction (PCR), suggested that expansion in GFSEC results in increased dedifferentiation compared to BEC. However, after 28 days of redifferentiation culture, morphology of tissue synthesized by GFSEC-expanded chondrocytes scored significantly higher on the Bern scale compared to BEC (6.4 +/- 0.3 points vs. 4.5 +/- 0.3 points in pellet culture and 6.0 +/- 0.4 points vs. 4.5 +/- 0.3 points on collagen-coated filters; p < 0.05). Expansion in GFSEC compared to BEC increased proteoglycan (PG) synthesis rate at day 9 (4.0-fold in pellet culture and 1.9-fold on collagen-coated filters; p < 0.01), PG release (6.7-fold in pellet culture and 3.2-fold on collagen-coated filters; p < 0.001), and final PG content at day 28 (1.6-fold in pellet culture and 1.5-fold on collagen-coated filters; p < 0.05). Redifferentiation on collagen-coated filters compared to pellet culture increased PG synthesis rate at day 9 (5.2-fold in BEC-expanded chondrocytes and 2.6-fold in GFSEC-expanded chondrocytes; p < 0.01), PG release (4.2-fold in BEC-expanded chondrocytes and 3.1-fold in GFSECexpanded chondrocytes; p < 0.01), and final PG content (1.3-fold in BEC-expanded chondrocytes and 1.9- fold in GFSEC-expanded chondrocytes; p < 0.01). Moreover, as visualized via electron microscopy, chondrocytes and organization of extracellular matrix cultured on filters was more similar to those found for hyaline cartilage. In conclusion, chondrocyte expansion in GFSEC and redifferentiation on collagen-coated filters resulted in most optimal chondrogenesis.

Anterior cruciate ligament (ACL) reconstruction surgery still has important problems to overcome, such as "donor site morbidity" and the limited choice of grafts in revision surgery. Tissue engineering of ligaments may provide a solution for these problems. Little is known about the optimal cell source for tissue engineering of ligaments. The aim of this study is to determine the optimal cell source for tissue engineering of the anterior cruciate ligament. Bone marrow stromal cells (BMSCs), ACL, and skin fibroblasts were seeded onto a resorbable suture material [poly(L-lactide/glycolide) multifilaments] at five different seeding densities, and cultured for up to 12 days. All cell types tested attached to the suture material, proliferated, and synthesized extracellular matrix rich in collagen type I. On day 12 the scaffolds seeded with BMSCs showed the highest DNA content (p < 0.01) and the highest collagen production (p < 0.05 for the two highest seeding densities). Scaffolds seeded with ACL fibroblasts showed the lowest DNA content and collagen production. Accordingly, BMSCs appear to be the most suitable cells for further study and development of tissue-engineered ligament.

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.

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.

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.

In recent years, the field of cartilage tissue engineering has seen a sharp increase in publications using many tissue engineering techniques and various analysis methods. Comparison between studies remains difficult, due to a lack of uniformity in methods used. A broad range of histological scoring systems is used to examine cartilage quality. Unfortunately, so far little is known on the reliability and correlation of these scoring systems. The objective of this study was to compare two frequently used cartilage repair grading scales, namely, the comprehensive O'Driscoll and the simple Pineda scale. We determined the intra- and interobserver variability of each score as well as the correlation between them. Thirty-eight joint section samples with variable cartilage quality were examined. Three observers documented their findings with both systems at two points in time. Statistical analysis showed very good intra- and interobserver reliability as well as a good correlation between the two scores. For the intraobserver variability of the O'Driscoll scale, we found an average difference of 0.05 with a SD of 0.93 in a 24-point score and a kappavalue of 0.87. For the interobserver reliability, the average difference was 0.001, SD 2.25, and a kappavalue of 0.92. The Pineda scale showed an average difference of 0.86 with a SD of 1.38 in a 14-point score and a kappavalue of 0.86 for the intraobserver reliability, whereas values for the interobserver reliability were average difference 0.82, SD 0.96, and a kappavalue of 0.89. The comparison between the two scales showed a high, inversely related correlation with a correlation coefficient of 0.71. From these results, we concluded that both the O'Driscoll and the Pineda scales are reliable semiquantitative cartilage scoring systems and that acceptance for general use of these two scores will benefit the reliability of literature on tissue engineering for cartilage repair. Thus, the added strength of comparison between published study results allows better understanding of cartilage repair publications and increases the impact of their results.