Tissue engineering, where one seeks cellular constructs capable of providing functional support for diseased or damaged tissues, can benefit from biomaterials that promote cell attachment, proliferation, and function. Multilayer nanofilms formed via LbL assembly are promising in this regard owing to their ease of formation and the possibility to tailor their chemical, mechanical, and biofunctional properties. The human liver represents a challenging but important tissue system upon which biomaterial-based regenerative strategies can be developed. While many studies attest to the utility of LbL assembled films in cell-contacting applications, little is known about their application to hepatocellular systems. Our intention here is to take the first steps toward multilayer nanofilm-based tissue engineering of the human liver.
We construct multilayer film biomaterials employing polysaccharides, polypeptides, and purely synthetic species proven to be successful in previous cell-contacting applications. As polysaccharides, chitosan (CHI), alginate (ALG), and a galactosylated chitosan (galCHI) are employed. The latter choice is motivated by studies showing hepatocytes to bind specifically to galactose moieties [43
]. As polypeptides, poly(L-lysine) (PLL) and poly(L-glutamic acid) (PGA) are employed. The PLL-PGA system has been quite extensively studied: chondrosarcoma cells have been shown to adhere to PLL terminated films more strongly than to glass or PGA terminated films [20
], and there is evidence of biocompatibility with SaOS-2 osteoblast-like cells for films ending with either PLL or PGA and with human periodontal ligament cells for films ending with PGA [47
]. As synthetic polymers, poly(allylamine hydrochloride) (PAH) and poly(styrene sulfonate) (PSS) are employed. Previous work on this system has shown films ending by PAH to improve adhesion of endothelial cells compared to protein coatings [48
], and films ending in PSS to support the attachment of primary hepatocytes from two month old rats [25
]. We consider films formed from a single “class” of polymer, as well as those formed from a polysaccharide and a polypeptide. Moreover, we modify the mechanical rigidity of certain films through a chemical cross-linking protocol.
Hypothesizing that useful biomaterials must at a minimum support cell attachment and growth, we begin by screening a number of candidate multilayer films against a human liver cell line: HepG2. Next, we employ films upon which HepG2 reach confluence, and investigate the attachment, growth, and function of two primary cell systems: adult rat hepatocytes and human fetal hepatoblasts. In doing so, we seek both to identify best performing systems; to understand the role of film composition, rigidity, biofunctionality, and charge on liver cell behavior; and to compare outcomes between HepG2 and ARH, systems commonly used in basic research studies, and HFHb, representing a viable cell source for human transplantation. Below, we summarize our finding in terms of these key variables.
We observe the type of polymers comprising the multilayer film biomaterial to play an important role in the cell response. As convincingly shown in , relatively few of the investigated polyelectrolyte pairs promote attachment and growth of the HepG2 cell line. Somewhat surprisingly, none of the pure polysaccharide films fared well, including those containing galactose units, previously shown to specifically enhance hepatocyte attachment [43
]. While the (PLL-ALG)n
-PLL system promoted strong attachment in all three hepatocellular systems, attachment on (PAH-PSS)n
by ARH was somewhat weaker than by HepG2 and HFHb, indicating a possible species specificity of this latter film. Film composition appears to play a large role in cell function as well: HFHb albumin production (in the absence of collagen) begins high and decreases about 30% over 8 days on the (PLL-PGA)n
-PLL film, begins low and increases 3 fold over 8 days on the (PAH-PSS)n
film, and exhibits a mid-time point maximum on the (PLL-ALG)n
film. The success of the PAH-PSS systems may be partially attributed to the known attraction between the side-chain styrene groups and hepatocytes [25
]. It is tempting to partially attribute the success of PLL-PGA and PLL-ALG systems to the presence of PLL. Indeed, PLL is known to strongly complex with oppositely charged polymers, a feature contributing to film rigidity (see below). However, based on the present study, it is difficult to definitively explain why the PLL-containing systems promote liver cell attachment and growth to a far greater extent than do other seemingly comparable systems.
Film terminal layer
Film terminal layer strongly influences the attachment and growth of HepG2 and, for certain systems, HFHb. In the case of HepG2, the two films observed to promote confluent layers, (PAH-PSS)n and (PLL-ALG)n-PLL-X, promote only sparse attachment when terminated with the opposite layer. Confluence is observed for HFHb i) on PLL-ALG films irrespective of terminal layer, ii) on PSS but not PAH terminated PAH-PSS films, iii) on PLL-PGA films irrespective of terminal layer in the presence of collagen, and iv) on PLL but not PGA terminated films in the absence of collagen. In contrast, film terminal layer appears to have little influence on the attachment of ARH.
While the importance of surface chemistry on the behavior of contacting cells has long been considered, the importance of mechanical properties has only been recognized more recently. In particular, cell behavior has been shown to depend sensitively on multilayer film rigidity: osteoblasts showed increased long term proliferation [28
], smooth muscle cells showed increased spreading [44
], and chondrosarcoma cells showed increased attachment [23
] on more rigid films. In this study, we seek to control film rigidity via EDC-NHS chemical cross-linking. We show, for the (PLL-ALG)n
-PLL system, HepG2 attachment and growth to be significantly enhanced by chemical cross-linking. However, no other system was positively affected in this way, including some films exhibiting appreciable HepG2 attachment/growth in the absence of cross-linking. In addition, it should be noted that the PAH-PSS system induces a comparable cell response to that of the cross-linked PLL-PGA and PLL-ALG systems, despite being significantly less rigid (in terms of QCMD measured mechanical moduli). Thus, factors beyond film rigidity are clearly at play here.
Overall, we observe the presence of biofunctional species (collagen I and IV) on top of the multilayer film biomaterial to influence the behavior of hepatocellular systems in only certain cases. For example, the presence of collagen is critical in promoting ARH attachment for all but one film considered. In contrast, collagen plays a significant role in promoting HFHb attachment in only two systems: confluence is reached in the presence of either rCI and hCIV on (PAH-PSS)n-PAH films at day 4 (but is not maintained through day 8), and confluence is maintained through day 8 in the presence of hCIV (but not rCI) in the (PLL-PGA)n-X system. These observations point to a species-specific difference in the role of adsorbed collagen on the biomaterial-cell interaction. Collagen influences the albumin production of HFHb for the PLL-PGA and PLL-ALG, but not the PAH-PSS, systems. Interestingly, collagen can inhibit albumin production at short times (PLL-PGA system), but generally enhances albumin production at longer times. It is possible that the degree of conformational change among adsorbed collagen molecules differs among the systems investigated here, and that these differences are partially behind our observed system-dependent collagen effect.
Overall film charge appears to be the least important variable for predicting cell behavior. For example, in the case of HepG2, of the two systems promoting significant cell attachment, one is positive and the other negative.
It is interesting to consider whether other film properties may play important roles in hepatocellular behavior. For example, film hydrophobicity is expected to vary among the systems considered here, with the general trend that chemical cross-linking [49
] and protein adsorption [50
] render hydrophilic surfaces (such as native polyelectrolyte multilayer films) more hydrophobic. Generally, cell attachment is weaker on more hydrophobic surfaces [51
], although the direct influence of hydrophobicity is difficult to distinguish from related properties, such as charge and tendency to adsorb protein [53
]. Within this framework, our finding that chemical cross-linking enhances cell attachment and growth seems to suggest increased film rigidity to more than offset any increased hydrophobicity. At the same time, our finding that collagen has a fairly modest influence on cell attachment and growth may suggest increased hydrophobicity to partially offset the presence of biofunctional species.
Through these studies, we identify multilayer biomaterial systems upon which HFHb systems reach confluence within a few days. A common requirement being a large number of transplanted cells, these systems become top candidates for various liver tissue engineering applications. Among systems promoting confluent HFHb layers, (PAH-PSS)n is appealing owing to its insensitivity to collagen and its promotion of a steadily increasing albumin secretion, a signal of healthy and functioning cells. Cross-linked (PLL-ALG)n and (PLL-PGA)n-PLL also appear to be promising, in part owing to their expected biodegradability