Dense collagen matrix accelerates osteogenic differentiation and rescues the apoptotic response to MMP inhibition☆
Introduction
The developmental biology of osteoblast differentiation and bone formation is well documented (reviewed [1]) and has culminated in the classic paradigms of mesenchymal condensation, followed by osteogenic differentiation for intramembranous ossification or the replacement of cartilage via vascular invasion for endochondral ossification [2]. The genetic strategies used to unpick the cascade of events that constitute osteoblast differentiation are not so well suited to fully reveal the role of osteoblast–matrix interactions in the process. Equally, the events of bone repair and regeneration are much less well understood, and follow a course that differs in some respects from that active during development [3].
Collagen is the major protein component of mature bone, and is the precursor matrix for direct bone repair; a priori it is a representative scaffold. The osteoblast senses the matrix through transmembrane collagen receptors of at least two classes — α1β1 and α2β1 integrins and discoidin domain receptors (DDR2) [4], providing outside→inside signalling. Mizuno et al. [5] have shown the importance of this integrin-mediated signalling for osteo-progenitor differentiation. The interaction with the matrix is not one way however, and the intracellular effects of receptor activation include the up-regulation of matrix metallo-proteinases (MMPs) [6] capable of remodelling the matrix and activating latent growth factors [7]. This in turn leads to the production of collagen fragments believed to be important for a second round of matrix–cell signalling (reviewed [8]). This is the classic dynamically reciprocal relationship of a cell and its ECM, and it is likely to have profound effects on the physical properties of any cell-containing scaffold. There is also a growing realisation that the physical characteristics of the matrix surrounding the cell – the elasticity of its microenvironment – can influence fate decisions of multipotential cells [9], possibly supplanting growth factor stimulation as a key determinant of osteoblast differentiation. The role of an osteoblast is to produce a stiff matrix, becoming an osteocyte in the process, the role of the osteocyte then being to sense and react to mechanical stimuli: one would therefore expect that this lineage is extremely sensitive to alterations in the stiffness of its environment.
It would be of great benefit to develop a scaffold that can act as a model for the study of basic biology of cells as well as a potential vehicle for cell delivery. The system investigated here is composed of type I collagen that has undergone plastic compression [10]. This physical process converts a hydrogel with less than 1% protein to a dense collagen (DC) matrix with approximately 13 wt.% protein, controlled fibrillar structure and significantly increased mechanical properties; effectively a workable soft tissue [10], [11], [12]. Concentrated collagen has a linear modulus of ~ 25 kPa, dense collagen has a linear modulus of ~ 1500 kPa [10]. This exceeds the rough estimate of osteoid elastic modulus, ~ 25 kPa [9].
The processing of cell-seeded collagen to form its dense counterpart requires the application of relatively large forces to the cells' framework; it has been shown that this is compatible with cell viability [10] but its effects on osteoblast differentiation have not been reported. The ability of osteoblasts to dramatically contract hydrated collagen (to half the original size in 24 h; [13]) has hitherto precluded the study of density (and hence stiffness) in a 3D setting. Therefore, to test the hypothesis that a stiffer 3D collagen matrix would promote osteoblast differentiation the current study examined how the rate of osteogenic differentiation was affected by the matrix using quantification of these markers, while the ultimate test of osteogenicity, the production of a functionally mineralised matrix, was also assessed. Finally, the role of matrix remodelling in the differentiation process was explored by targeting MMP activity.
Section snippets
Isolation of calvarial osteoblasts
Cells were isolated from the calvaria of neonatal mice (ICR-CD1) at P5 [14], based on the original method [15]. In brief, sequential digests with crude Type IA collagenase (Sigma) were used on pooled calvaria (from 10–20 pups), those cells being released first were discarded and subsequent fractions (up to 4) were collected and pooled. Cells were maintained and expanded for a maximum of 2 passages and cultured in LG DMEM (Invitrogen), 10% FBS (PAA), p/s (PAA) and ascorbate-2-phosphate
Results
The dense collagen cultures seeded with primary mouse calvarial osteoblasts, in control medium (ascorbate only), osteogenic medium (ascorbate plus β-glycerophosphate) and dex medium — as osteogenic with the addition of dexamethasone, were assessed after 7 and 14 days. The expression of 5 genes was analysed by Q-PCR: bone sialoprotein (Bsp); alkaline phosphatase (Alp); Runx2; Col1a1 and Mmp-13. The basal levels of expression are given in Fig. 1A demonstrating that there are relatively low
Discussion
From the gene expression profiles described it is evident that as a whole the population has transitioned from pre-osteoblast to osteoblast [19], [25]. The process by which osteoblasts undergo the transition to osteocytes during development of the skeleton is still contentious. Little is known of how it may occur during osseous wound repair. The evidence indicates that embedded pre-osteoblasts rapidly differentiate and then behave as static osteoblasts [26], mineralising the matrix in a
Conclusion
The potential of the matrix (or scaffold) to promote regeneration has long been recognised. It is now evident that these studies will have implications for the understanding of basic biology, by revealing new influences and allowing the control of novel variables. By producing a native matrix scaffold that mimics the density (and hence stiffness) of osteoid, it has been possible for the first time, to characterise the differentiation of primary pre-osteoblasts as it may occur in vivo. Symmetry
References (40)
- et al.
Mammalian collagen receptors
Matrix Biol
(2007) - et al.
Matrix metalloproteinase-dependent activation of latent transforming growth factor-beta controls the conversion of osteoblasts into osteocytes by blocking osteoblast apoptosis
J Biol Chem
(2002) - et al.
Matrix remodeling during endochondral ossification
Trends Cell Biol
(2004) - et al.
Matrix elasticity directs stem cell lineage specification
Cell
(2006) - et al.
Molecular-cloning and expression of collagenase-3, a novel human matrix metalloproteinase produced by breast carcinomas
J Biol Chem
(1994) - et al.
Bone-related gene profiles in developing calvaria
Gene
(2006) - et al.
Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation
Cell
(1997) - et al.
Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts
Cell
(1997) - et al.
Cell lines and primary cell cultures in the study of bone cell biology
Mol Cell Endocrinol
(2004) - et al.
Hedgehog stimulates only osteoblastic differentiation of undifferentiated KS483 cells
Bone
(2003)
Mechanical strain induces collagenase-3 (MMP-13) expression in MC3T3-E1 osteoblastic cells
J Biol Chem
Metalloproteinase shedding of Fas ligand regulates beta-amyloid neurotoxicity
Curr Biol
The complexities of skeletal biology
Nature
Divide, accumulate, differentiate: cell condensation in skeletal development revisited
Int J Dev Biol
Fracture healing as a post-natal developmental process: molecular, spatial, and temporal aspects of its regulation
J Cell Biochem
Type I collagen-induced osteoblastic differentiation of bone-marrow cells mediated by collagen-alpha 2 beta 1 integrin interaction
J Cell Physiol
Expression of matrix metalloproteinases during ascorbate-induced differentiation of osteoblastic MC3T3-E1 cells
J Bone Min Res
Ultrarapid engineering of biomimetic materials and tissues: Fabrication of nano- and microstructures by plastic compression
Adv Funct Mat
Use of multiple unconfined compression for control of collagen gel scaffold density and mechanical properties
Soft Matter
Effect of multiple unconfined compression on cellular dense collagen scaffolds for bone tissue engineering
J Mat Sci-Mat Med
Cited by (70)
Harnessing the dental cells derived from human induced pluripotent stem cells for hard tissue engineering
2023, Journal of Advanced ResearchCombining sclerostin neutralization with tissue engineering: An improved strategy for craniofacial bone repair
2022, Acta BiomaterialiaCitation Excerpt :In addition, neural-crest derived osteogenic cells are known to be more efficient in osteoblast differentiation and bone repair than their mesoderm counterparts [78]. Regarding the use of dense collagen hydrogels as a scaffold, we and others have previously demonstrated that such scaffolds allowed the addition of MSC and a fiber density favoring osteogenesis, while being perfectly tolerated by the host upon implantation [39,44,52,55,56,58,79]. In our study, the addition of DPSC within the dense collagen hydrogels markedly improved bone regeneration in WT mice, which is consistent with previous studies in rodent models [39,43,44,56,73].
Dense collagen-based scaffolds for soft tissue engineering applications
2021, Tissue Engineering Using Ceramics and Polymers, Third EditionAcellular dense collagen-S53P4 bioactive glass hybrid gel scaffolds form more bone than stem cell delivered constructs
2021, Materials Science and Engineering CFabrication and characterization of collagen-based injectable and self-crosslinkable hydrogels for cell encapsulation
2018, Colloids and Surfaces B: Biointerfaces
- ☆
This work was supported by UCLH CRDC grants to S.N.N. and P.G.B.