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In-vivo generation of bone via endochondral ossification by in-vitro chondrogenic priming of adult human and rat mesenchymal stem cells.

Farrell E, Both SK, Odörfer KI, Koevoet W, Kops N, O'Brien FJ, Baatenburg de Jong RJ, Verhaar JA, Cuijpers V, Jansen J, Erben RG, van Osch GJ - BMC Musculoskelet Disord (2011)

Bottom Line: Thereby we found that osteoblasts in the bone were almost entirely of host origin but the osteocytes are of both host and donor origin.Furthermore, addition of β-glycerophosphate to the chondrogenic medium did not hamper this process.In conclusion these data indicate that in-vitro chondrogenic differentiation of human MSCs could lead to an alternative and superior approach for bone tissue engineering.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Orthopaedics, Erasmus MC University Medical Centre Rotterdam, The Netherlands. eric.farrell@nuigalway.ie

ABSTRACT

Background: Bone grafts are required to repair large bone defects after tumour resection or large trauma. The availability of patients' own bone tissue that can be used for these procedures is limited. Thus far bone tissue engineering has not lead to an implant which could be used as alternative in bone replacement surgery. This is mainly due to problems of vascularisation of the implanted tissues leading to core necrosis and implant failure. Recently it was discovered that embryonic stem cells can form bone via the endochondral pathway, thereby turning in-vitro created cartilage into bone in-vivo. In this study we investigated the potential of human adult mesenchymal stem cells to form bone via the endochondral pathway.

Methods: MSCs were cultured for 28 days in chondrogenic, osteogenic or control medium prior to implantation. To further optimise this process we induced mineralisation in the chondrogenic constructs before implantation by changing to osteogenic medium during the last 7 days of culture.

Results: After 8 weeks of subcutaneous implantation in mice, bone and bone marrow formation was observed in 8 of 9 constructs cultured in chondrogenic medium. No bone was observed in any samples cultured in osteogenic medium. Switch to osteogenic medium for 7 days prevented formation of bone in-vivo. Addition of β-glycerophosphate to chondrogenic medium during the last 7 days in culture induced mineralisation of the matrix and still enabled formation of bone and marrow in both human and rat MSC cultures. To determine whether bone was formed by the host or by the implanted tissue we used an immunocompetent transgenic rat model. Thereby we found that osteoblasts in the bone were almost entirely of host origin but the osteocytes are of both host and donor origin.

Conclusions: The preliminary data presented in this manuscript demonstrates that chondrogenic priming of MSCs leads to bone formation in vivo using both human and rat cells. Furthermore, addition of β-glycerophosphate to the chondrogenic medium did not hamper this process. Using transgenic animals we also demonstrated that both host and donor cells played a role in bone formation. In conclusion these data indicate that in-vitro chondrogenic differentiation of human MSCs could lead to an alternative and superior approach for bone tissue engineering.

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Osteogenic culture or switch prevents endochondral ossification but addition of β-glycerophosphate does not. Representative hematoxilin-eosin stained slides of implanted pellets in immune deficient mice for 8 weeks. Primed chondrogenically bone, cartilage and marrow stroma are visible (Ai). For the switch 1 condition the chondrogenic medium was replaced during the last 7 days for osteogenic medium which resulted in cartilage-like tissue in the inside and undefined tissue on the outside (Bi). For the switch 2 condition β-glycerophosphate was added during the last 7 days of culture and bone, cartilage and marrow stroma are observed (Ci). When the chondrogenic primed pellets were implanted for 14 weeks only bone and marrow stroma were visible. For quantitative analysis all pictures were pseudo colored, red (bone), blue (marrow stroma) green (cartilage), undefined tissue (yellow) (Aii, Bii, Cii, Dii). Figure 2, E and F show Safranin O staining of in vitro chondrogenically cultured pellets retrieved after 8 week in vivo. Weakly positive staining demonstrates the presence of glycosaminoglycans within a cartilage matrix being degraded to make way for bone and marrow formation which surrounds the remnants of the cartilage matrix.
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Figure 2: Osteogenic culture or switch prevents endochondral ossification but addition of β-glycerophosphate does not. Representative hematoxilin-eosin stained slides of implanted pellets in immune deficient mice for 8 weeks. Primed chondrogenically bone, cartilage and marrow stroma are visible (Ai). For the switch 1 condition the chondrogenic medium was replaced during the last 7 days for osteogenic medium which resulted in cartilage-like tissue in the inside and undefined tissue on the outside (Bi). For the switch 2 condition β-glycerophosphate was added during the last 7 days of culture and bone, cartilage and marrow stroma are observed (Ci). When the chondrogenic primed pellets were implanted for 14 weeks only bone and marrow stroma were visible. For quantitative analysis all pictures were pseudo colored, red (bone), blue (marrow stroma) green (cartilage), undefined tissue (yellow) (Aii, Bii, Cii, Dii). Figure 2, E and F show Safranin O staining of in vitro chondrogenically cultured pellets retrieved after 8 week in vivo. Weakly positive staining demonstrates the presence of glycosaminoglycans within a cartilage matrix being degraded to make way for bone and marrow formation which surrounds the remnants of the cartilage matrix.

Mentions: Following the results observed in donors 1 and 2, the osteogenic condition was discontinued with donor 3. In addition to the complete switch to serum containing osteogenic medium for the last culture week, samples were maintained on chondrogenic medium with β-glycerophosphate to achieve mineralisation of the matrix as observed previously [6]. This experiment was performed with cell-seeded scaffolds and pellet cultures. Bone formation was observed in all pellets that were primed chondrogenically (Figure 2Ai) confirming results of the experiments with scaffolds. Once again, under the initial switch conditions of culturing cells for 1 week in osteogenic medium after 3 weeks in chondrogenic medium, no endochondral ossification or bone formation was observed (Figure 2Bi). Only one of the three implanted pellets of the second switch condition where β-glycerophosphate was added to the chondrogenic medium, was retrieved. Interestingly, in this condition bone formation was observed to occur similarly to the chondrogenic condition (Figure 2Ci). Once again, a marrow stroma was also observed within the pellets around the area of bone formation. This effect was also observed in all 3 scaffold samples that were cultured under identical conditions in the rat study. Addition of β-glycerophosphate did not prevent bone formation in vivo. Histomorphometry (Figure 2Aii, Bii, Cii) revealed that in the pellet constructs with bone formation 32 ± 10% of the construct area consisted of bone tissue, and 37 ± 24% of bone marrow. The rest of the area (39 ± 27%) was cartilage as confirmed by positive collagen type II immunohistochemistry. This data is presented in Table 1. Safranin O staining of these pellets showed the presence of small amounts of GAGs remaining in the cartilage like matrix. This indicates that in this phase most proteoglycans have been degraded, a process which occurs during endochondral ossification. Figures 2E and 2F demonstrate all stages of endochondral ossification in the same pellet, cartilage degradation, blood vessel invasion and bone and marrow formation.


In-vivo generation of bone via endochondral ossification by in-vitro chondrogenic priming of adult human and rat mesenchymal stem cells.

Farrell E, Both SK, Odörfer KI, Koevoet W, Kops N, O'Brien FJ, Baatenburg de Jong RJ, Verhaar JA, Cuijpers V, Jansen J, Erben RG, van Osch GJ - BMC Musculoskelet Disord (2011)

Osteogenic culture or switch prevents endochondral ossification but addition of β-glycerophosphate does not. Representative hematoxilin-eosin stained slides of implanted pellets in immune deficient mice for 8 weeks. Primed chondrogenically bone, cartilage and marrow stroma are visible (Ai). For the switch 1 condition the chondrogenic medium was replaced during the last 7 days for osteogenic medium which resulted in cartilage-like tissue in the inside and undefined tissue on the outside (Bi). For the switch 2 condition β-glycerophosphate was added during the last 7 days of culture and bone, cartilage and marrow stroma are observed (Ci). When the chondrogenic primed pellets were implanted for 14 weeks only bone and marrow stroma were visible. For quantitative analysis all pictures were pseudo colored, red (bone), blue (marrow stroma) green (cartilage), undefined tissue (yellow) (Aii, Bii, Cii, Dii). Figure 2, E and F show Safranin O staining of in vitro chondrogenically cultured pellets retrieved after 8 week in vivo. Weakly positive staining demonstrates the presence of glycosaminoglycans within a cartilage matrix being degraded to make way for bone and marrow formation which surrounds the remnants of the cartilage matrix.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3045394&req=5

Figure 2: Osteogenic culture or switch prevents endochondral ossification but addition of β-glycerophosphate does not. Representative hematoxilin-eosin stained slides of implanted pellets in immune deficient mice for 8 weeks. Primed chondrogenically bone, cartilage and marrow stroma are visible (Ai). For the switch 1 condition the chondrogenic medium was replaced during the last 7 days for osteogenic medium which resulted in cartilage-like tissue in the inside and undefined tissue on the outside (Bi). For the switch 2 condition β-glycerophosphate was added during the last 7 days of culture and bone, cartilage and marrow stroma are observed (Ci). When the chondrogenic primed pellets were implanted for 14 weeks only bone and marrow stroma were visible. For quantitative analysis all pictures were pseudo colored, red (bone), blue (marrow stroma) green (cartilage), undefined tissue (yellow) (Aii, Bii, Cii, Dii). Figure 2, E and F show Safranin O staining of in vitro chondrogenically cultured pellets retrieved after 8 week in vivo. Weakly positive staining demonstrates the presence of glycosaminoglycans within a cartilage matrix being degraded to make way for bone and marrow formation which surrounds the remnants of the cartilage matrix.
Mentions: Following the results observed in donors 1 and 2, the osteogenic condition was discontinued with donor 3. In addition to the complete switch to serum containing osteogenic medium for the last culture week, samples were maintained on chondrogenic medium with β-glycerophosphate to achieve mineralisation of the matrix as observed previously [6]. This experiment was performed with cell-seeded scaffolds and pellet cultures. Bone formation was observed in all pellets that were primed chondrogenically (Figure 2Ai) confirming results of the experiments with scaffolds. Once again, under the initial switch conditions of culturing cells for 1 week in osteogenic medium after 3 weeks in chondrogenic medium, no endochondral ossification or bone formation was observed (Figure 2Bi). Only one of the three implanted pellets of the second switch condition where β-glycerophosphate was added to the chondrogenic medium, was retrieved. Interestingly, in this condition bone formation was observed to occur similarly to the chondrogenic condition (Figure 2Ci). Once again, a marrow stroma was also observed within the pellets around the area of bone formation. This effect was also observed in all 3 scaffold samples that were cultured under identical conditions in the rat study. Addition of β-glycerophosphate did not prevent bone formation in vivo. Histomorphometry (Figure 2Aii, Bii, Cii) revealed that in the pellet constructs with bone formation 32 ± 10% of the construct area consisted of bone tissue, and 37 ± 24% of bone marrow. The rest of the area (39 ± 27%) was cartilage as confirmed by positive collagen type II immunohistochemistry. This data is presented in Table 1. Safranin O staining of these pellets showed the presence of small amounts of GAGs remaining in the cartilage like matrix. This indicates that in this phase most proteoglycans have been degraded, a process which occurs during endochondral ossification. Figures 2E and 2F demonstrate all stages of endochondral ossification in the same pellet, cartilage degradation, blood vessel invasion and bone and marrow formation.

Bottom Line: Thereby we found that osteoblasts in the bone were almost entirely of host origin but the osteocytes are of both host and donor origin.Furthermore, addition of β-glycerophosphate to the chondrogenic medium did not hamper this process.In conclusion these data indicate that in-vitro chondrogenic differentiation of human MSCs could lead to an alternative and superior approach for bone tissue engineering.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Orthopaedics, Erasmus MC University Medical Centre Rotterdam, The Netherlands. eric.farrell@nuigalway.ie

ABSTRACT

Background: Bone grafts are required to repair large bone defects after tumour resection or large trauma. The availability of patients' own bone tissue that can be used for these procedures is limited. Thus far bone tissue engineering has not lead to an implant which could be used as alternative in bone replacement surgery. This is mainly due to problems of vascularisation of the implanted tissues leading to core necrosis and implant failure. Recently it was discovered that embryonic stem cells can form bone via the endochondral pathway, thereby turning in-vitro created cartilage into bone in-vivo. In this study we investigated the potential of human adult mesenchymal stem cells to form bone via the endochondral pathway.

Methods: MSCs were cultured for 28 days in chondrogenic, osteogenic or control medium prior to implantation. To further optimise this process we induced mineralisation in the chondrogenic constructs before implantation by changing to osteogenic medium during the last 7 days of culture.

Results: After 8 weeks of subcutaneous implantation in mice, bone and bone marrow formation was observed in 8 of 9 constructs cultured in chondrogenic medium. No bone was observed in any samples cultured in osteogenic medium. Switch to osteogenic medium for 7 days prevented formation of bone in-vivo. Addition of β-glycerophosphate to chondrogenic medium during the last 7 days in culture induced mineralisation of the matrix and still enabled formation of bone and marrow in both human and rat MSC cultures. To determine whether bone was formed by the host or by the implanted tissue we used an immunocompetent transgenic rat model. Thereby we found that osteoblasts in the bone were almost entirely of host origin but the osteocytes are of both host and donor origin.

Conclusions: The preliminary data presented in this manuscript demonstrates that chondrogenic priming of MSCs leads to bone formation in vivo using both human and rat cells. Furthermore, addition of β-glycerophosphate to the chondrogenic medium did not hamper this process. Using transgenic animals we also demonstrated that both host and donor cells played a role in bone formation. In conclusion these data indicate that in-vitro chondrogenic differentiation of human MSCs could lead to an alternative and superior approach for bone tissue engineering.

Show MeSH
Related in: MedlinePlus