Post microtextures accelerate cell proliferation and osteogenesis

Abstract

The influence of surface microtexture on osteogenesis was investigated in vitro by examining the proliferation and differentiation characteristics of a class of adult stem cells and their progeny, collectively known as connective tissue progenitor cells (CTPs). Human bone marrow-derived CTPs were cultured for up to 60 days on smooth polydimethylsiloxane (PDMS) surfaces and on PDMS with post microtextures that were 10 microm in diameter and 6 microm in height, with 10 microm separation. DNA quantification revealed that the numbers of CTPs initially attached to both substrates were similar. However, cells on microtextured PDMS transitioned from lag phase after 4 days of culture, in contrast to 6 days for cells on smooth surfaces. By day 9 cells on the smooth surfaces exhibited arbitrary flattened shapes and migrated without any preferred orientation. In contrast, cells on the microtextured PDMS grew along the array of posts in an orthogonal manner. By days 30 and 60 cells grew and covered all surfaces with extracellular matrix. Western blot analysis revealed that the expression of integrin alpha5 was greater on the microtextured PDMS compared with smooth surfaces. Real time reverse transcription-polymerase chain reaction revealed that gene expression of alkaline phosphatase had decreased by days 30 and 60, compared with that on day 9, for both substrates. Gene expression of collagen I and osteocalcin was consistently greater on post microtextures relative to smooth surfaces at all time points.

Fig. 1

Qualitative illustration of the three major periods of cell and tissue development in bone formation [14,15]. Relationship between cell growth and differentiation-related gene expression during the in vitro cultivation of osteoblast progenitor cells. Three stages of maturation are reflected by maximal expression of growth and differentiation markers. At the early stage, osteoblast progenitor cells synthesize significant levels of Col I to support matrix formation. Although Col I levels are highest during the proliferation stage, matrix deposition continues to increase throughout the entire culture period. The termination of growth and accumulated extracellular matrix (ECM) accelerates upregulation of AP, an early stage marker of osteogenesis. OC is expressed towards the end of the proliferation stage, during development of the osteoblast phenotype, and reaches peak levels during mineralization. The timescales are derived from data from human mesen-chymal stem cells (MSCs) which were culture expanded. In contrast, connective tissue progenitor (CTP) cells are primary bone marrow-derived cells and the corresponding timescale for osteogensis is not known. AP, alkaline phosphatase; Col I, collagen type I; OC, osteocalcin.

Fig. 2

SEM images of PDMS substrates and CTPs on PDMS post microtextures and control surfaces on days 9, 30 and 60. The PDMS substrates were produced by soft lithography techniques. (a) PDMS post microtextures 6 µm in height and 10 µm in diameter, with 10 µm separation between posts. (b) Control (smooth PDMS) surface. (c) CTPs attached to post microtextures and control surfaces with varying cell morphologies. On post microtextures on day 9 CTPs mostly tended to attach next to the posts and spread between them while directing their processes towards posts and other cells. On day 30 we can see increased cell growth on the post microtexture scaffolds. By day 60 numerous cells had grown and spread over the top of the post microtextures and covered most of surface with ECM. In contrast, cells on the control surfaces exhibited arbitrary flattened shapes and migrated without any preferred orientation for up to 60 days.

Fig. 3

Time dependent (a) growth curves and (b) fold changes of CTPs attached to PDMS substrates. DNA quantification analysis revealed that the number of CTPs initially attached to PDMS substrates were almost identical. However, the greatest fold change was observed between day 4 and day 5, as the cells on PDMS post microtextures were about to exit from lag phase. Cells on the control surfaces were about to exit from lag phase on days 6 and 7. After exiting from the lag phase, the cell growth curves were similar on PDMS post microtextures and control surfaces.

Fig. 4

CTP number on PDMS post microtextures and corresponding control surfaces. On day 9 the number of CTPs on the post microtextures was greater than on the control surfaces. The post microtextures exhibited a significant increase in CTPs on day 30. On day 60 the number of CTPs increased on post microtextures compared with the control surfaces. Fluorescent images show cell nuclei stained with DAPI and reveal more cells on post microtextures than control surfaces. (Note: the original color images were converted to grayscale and reversed to provide visual clarity). The PicoGreen DNA quantification was performed a total of nine times (three replicates for each of the three patients, i.e. n = 9 for each substrate) as per our standard laboratory protocol. ^*Statistically significant compared with control surfaces on day 30 (P < 0.05).

Fig. 5

Integrins and GAPDH expression in CTPs after 30 and 60 days culture on PDMS post microtxtures and control surfaces. Integrins α1, α2, α5 and β1 were expressed by the cells on all surfaces, although integrin α1 was expressed at generally lower levels. On days 30 and 60, integrins α1, α2, and β1 showed comparable expression levels between post microtextures and smooth surfaces. In contrast, integrin α5 exhibited greater expression level on the PDMS post microtextures compared with smooth surfaces.

Fig. 6

Gene expression of (a) AP, (b) collagen type I (Col I) and (c) osteocalcin (OC) by CTPs after 9, 30 and 60 day on PDMS post microtextures and control surfaces. (a) AP mRNA expression during development, with the highest levels present on day 9 on post microtextures, while it was expressed more highly on the control surface on day 60. Fluorescent images show cells on the post microtextures stained more intensely for AP compared with control surfaces on day 9, and AP increased on all surfaces by days 30 and 60. (b) mRNA of Col I expression at all time points, with slightly higher expression on day 30 and significantly higher on day 60. (c) Compared with day 9, OC mRNA expression showed a significant increase in cells grown on post microtextures on day 60 compared with cells grown on the control surface. Phase contrast images show von Kossa stain and the intensity of this stain on post microtextures increased with time compared with the control surfaces. The real time RT-PCR was performed a total of nine times (three replicates for each of the three patients, i.e. n = 9 per substrate) as per our standard laboratory protocols. ^*Statistically significant compared with control surfaces on same day (P < 0.05).

Fig. 6

Gene expression of (a) AP, (b) collagen type I (Col I) and (c) osteocalcin (OC) by CTPs after 9, 30 and 60 day on PDMS post microtextures and control surfaces. (a) AP mRNA expression during development, with the highest levels present on day 9 on post microtextures, while it was expressed more highly on the control surface on day 60. Fluorescent images show cells on the post microtextures stained more intensely for AP compared with control surfaces on day 9, and AP increased on all surfaces by days 30 and 60. (b) mRNA of Col I expression at all time points, with slightly higher expression on day 30 and significantly higher on day 60. (c) Compared with day 9, OC mRNA expression showed a significant increase in cells grown on post microtextures on day 60 compared with cells grown on the control surface. Phase contrast images show von Kossa stain and the intensity of this stain on post microtextures increased with time compared with the control surfaces. The real time RT-PCR was performed a total of nine times (three replicates for each of the three patients, i.e. n = 9 per substrate) as per our standard laboratory protocols. ^*Statistically significant compared with control surfaces on same day (P < 0.05).