Peptides are the next step along this surface chemistry driven pathway, allowing biomaterials to mimic natural ECM, avoiding some of the risk factors of using whole proteins from either xenological or human sources. This work has enabled researchers to establish the origins of important signals in the differentiation pathway of stem cells. This is being conducted using several novel screening methods, including the expression of peptides in a
Page 24 bacterial model where the bacterium are transfected with a plasmid containing the coding for different peptides, which are then expressed in the bacteria69.
Peptides that are engineered synthetically are very useful as a research tool as they simulate the ECM in a controllable manner. It is possible to distinguish the differing roles of the various molecules in ECM by engineering the individual peptides and testing them in isolation70. Isolating the peptides was an important step towards determining the individual capacity of the peptides and the peptides which were relevant for the osteogenic lineage. More established RGD and BMP were determined to be very relevant for the osteogenic lineage. During one study peptide amphiphiles were functionalised with RGD and DGEA which were seen to be osteoinductive in their properties71. Both of the peptides showed an increase in osteoinductivity when cultured with media containing growth factors, whilst RGD showed a degree of phenotypic change when cultured in the absence of growth factors. This demonstrated very clearly the RGD has osteoinductive qualities that may be used to functionalise biomaterial surfaces71.
The osteoinductive properties of the RGD peptide are difficult to deny. The only foreseeable problems (which are considerable) with the use of peptides on a large scale is cost and availability. It is very expensive and time consuming to produce peptides, and if they were to be used commercially would be required in large volumes. The expense of using peptides is their limiting factor, and for this reason, identifying active molecules to bind material surfaces is progressing as the analytical techniques have improved. Isolating the chemical groups active from within the peptide, and applying them to biomaterial surfaces is a new strategy within the field. This is more cost effective than using peptides, and is starting to yield similar results, in vitro.
Page 25 1.18 Chemical Modification
There are many methods of modifying a polymer surface72. A polymer surface usually needs to be functionalised before the application of an active chemical group. There are several ways to do this depending on the outcome that is required. To achieve maximum functionalization, a polyfunctional agent can be grafted to the surface of the polymer. This allows the more functional units to be available and effectively increases the functional units per given unit of surface area compared to that of using a single function molecule. However, there can be problems with steric hindrance when functional groups are tightly packed together on a surface. One way to overcome this difficulty is to use a spacer molecule, which allows movement and often acts as a protective layer to keep the active chemical group away from hydrophobic surfaces, which can denature some bioactive compounds73. The chain length of these spacer molecules is variable, and finding an optimum chain length for a particular chemical group is important and could be a reason for the conflicting results reported in the literature where surface modifications appear to have the same chemical terminal group but induce different differentiation pathways in stem cells74.
By using combinations of functional groups arranged in different ways it is possible to mimic the natural ECM in various tissues. The chemical groups that are of particular interest for skeletal regeneration are -NH2, CH3, -OH and –COOH as all these groups are found on bone ECM.
Page 26 1.19 Amine Groups
Amine groups have been shown to play an active role in the immobilisation of proteins, because of their positive charge and it is thought that the amine groups have the capacity to attract and interact with proteins on the surface of a biomaterial75. However there have been some variable results reported in terms of the effects observed on MSCs exposed to these modifications, in some cases have been directly contradictory. Amine rich surfaces have been reported to be osteogenic,62 chondrogenic65 and non-differentiating75. Clearly the reaction of the cells to the terminal groups is only part of the differentiation and presentation of the modification is likely to also play a role. Interestingly, there have been differences seen when the same chemical is deposited onto a biomaterials surface in different ways (e.g.
APTES). APTES was deposited using a Plasma technique76 showed no significant increase in osteogenic response with a pre-osteoblastic cell line compared to a control untreated substrate, where-as APTES applied using a wet chemical technique caused an osteogenic effect from MSCs62. This contradictory data merits further investigation, as it appears that amine groups if presented in the correct way could have powerful osteogenic properties. It is essential to isolate the parameters that affect the presentation of the amine and determine which of the many possible modification techniques will have the most clinical relevance.
Clearly there is need for clarification of the surface modifications at a molecular level. Clarification may come from varying presentation of the terminal groups to achieve an optimised and reproducible response from the cells.