**4. Differentiation of VSMCs cultured in honeycombs**

Caldesmon is an actin-linked regulatory protein, and caldesmon heavy chain (h-CaD) is expressed abundantly and specifically in contractile VSMCs. h-CaD localizes to the actomyosin contractile structure in VSMCs as an integral component of smooth muscle thin filaments [18]. Caldesmon also works as a potent repressor of cancer cell invasion because its ectopic expression reduces the number of podosomes/invadopodia and decreases extracellular matrix degradation. The depletion of caldesmon facilitates the formation of podosomes/invadopodia and cell invasion [19]. As VSMCs cultured in honeycombs for 14 days express h-CaD [4, 6], these cells could be classified to the contractile phenotype.

Some other cells also differentiate in honeycombs. By usage of honeycomb scaffolds, rat axonal regeneration [20], differentiation of pluripotent embryonic stem (ES) cell-derived embryoid bodies (EBs) into hepatocyte-like cells [21], and differentiation of mesenchymal stem cells into osteoblasts [22] have been reported. For example, EBs formed from ES cells become liverspecific gene-positive and albumin-positive cells, and they form cord-like structures in honeycombs that are not present in two-dimensional monolayer culture systems [21]. When a honeycomb scaffold including EB-derived hepatocyte-like cells is transplanted into the median lobes of partially hepatectomized nude mice, cells that are positive for both albumin and cytokeratin 18 appear in the transplant and form clustered aggregates after 7 and 14 days. These data suggest that a honeycomb is a useful scaffold culture system for regulation of cellular differentiation and for advanced tissue engineering.

Three-Dimensional "Honeycomb" Culture System that Helps to Maintain the Contractile Phenotype of… http://dx.doi.org/10.5772/60960 449

OAZ1; lane 3, pTracer-OAZ∆T205∆AUG2, which expresses 29-kDa OAZ1. After transfection, VSMCs were collected at 24 h, a cell extract was prepared, and Western blot analysis was performed. (b) Degree of inhibition of ODC by OAZ1 was measured. The activity of OAZ1 was determined as an inhibitory percentage of ODC activity of the extract from ODC-overproducing FM3A (EXOD-1) cells. ●, extract from VSMCs expressing 24.5-kDa OAZ1; ▲, extract from VSMCs expressing 29- and 24.5-kDa OAZ1; and ○, extract from VSMCs expressing 29-kDa OAZ1. Values are means of duplicate determinations. (B) Inhibition of ODC activity using extracts from VSMCs cultured on plates and in honey‐ combs. OAZ1 activity was determined as an inhibitory percentage of ODC activity of the extract from EXOD-1 cells. Values are means ± standard deviation (SD) of triplicate determinations. (C) Western blot analysis of OAZ1, ODC, ty‐ rosine-phosphorylated FAK (pY-FAK), α-actin, myosin heavy chain, and β-actin of VSMCs and ODC-VSMCs cultured on plates and in honeycombs. P, EXOD-1 cells as a positive control. Relative intensity on day 7 was quantified. The intensity of OAZ1 was quantified as the sum of the 29- and 24.5-kDa bands. ND, not detectable. Values are means ± SD of triplicate determinations. (D) OAZ1 degradation. ODC-VSMCs cultured on plates and in honeycombs for 3 days

VSMC proliferation is reportedly up-regulated via an increase in the stability of S-phase kinaseassociated protein-2, E3 ubiquitin protein ligase (SKP2) by autophosphorylation of FAK-Tyr397 [17]. In ODC-VSMCs cultured on plates, the increased rate of proliferation is accompanied by an increase of phosphorylated FAK. Interestingly, when the effect of spermine on FAK autophosphorylation *in vitro* was investigated, FAK phosphorylation was stimulated by spermine [15]. The low levels of intracellular polyamines in VSMCs cultured in honeycombs could inhibit the autophosphorylation of FAK, and this may contribute to the proliferative

Caldesmon is an actin-linked regulatory protein, and caldesmon heavy chain (h-CaD) is expressed abundantly and specifically in contractile VSMCs. h-CaD localizes to the actomyosin contractile structure in VSMCs as an integral component of smooth muscle thin filaments [18]. Caldesmon also works as a potent repressor of cancer cell invasion because its ectopic expression reduces the number of podosomes/invadopodia and decreases extracellular matrix degradation. The depletion of caldesmon facilitates the formation of podosomes/invadopodia and cell invasion [19]. As VSMCs cultured in honeycombs for 14 days express h-CaD [4, 6],

Some other cells also differentiate in honeycombs. By usage of honeycomb scaffolds, rat axonal regeneration [20], differentiation of pluripotent embryonic stem (ES) cell-derived embryoid bodies (EBs) into hepatocyte-like cells [21], and differentiation of mesenchymal stem cells into osteoblasts [22] have been reported. For example, EBs formed from ES cells become liverspecific gene-positive and albumin-positive cells, and they form cord-like structures in honeycombs that are not present in two-dimensional monolayer culture systems [21]. When a honeycomb scaffold including EB-derived hepatocyte-like cells is transplanted into the median lobes of partially hepatectomized nude mice, cells that are positive for both albumin and cytokeratin 18 appear in the transplant and form clustered aggregates after 7 and 14 days. These data suggest that a honeycomb is a useful scaffold culture system for regulation of

were treated with 20 µg/mL cycloheximide for the indicated time. Data adapted from reference 15.

**4. Differentiation of VSMCs cultured in honeycombs**

these cells could be classified to the contractile phenotype.

cellular differentiation and for advanced tissue engineering.

inhibition of VSMCs.

448 Muscle Cell and Tissue

Figure 5. Effect of the transient expression of OAZ1 in ODC-VSMCs cultured on plates and in honeycombs. After ODC-VSMCs were transfected with pTracer-OAZ∆T205∆AUG1 (24.5-kDa OAZ1), the cells were cultured as shown **Figure 5.** Effect of the transient expression of OAZ1 in ODC-VSMCs cultured on plates and in honeycombs. After ODC-VSMCs were transfected with pTracer-OAZΔT205ΔAUG1 (24.5-kDa OAZ1), the cells were cultured as shown in (A). Transfected ODC-VSMCs were then cultured either on plates or in honeycombs for 1, 3, and 7 days. (B) Cell num‐ ber on days 1 and 3. (C) Western blot analysis of OAZ1, ODC, pY-FAK, α-actin, myosin heavy chain, and β-actin. Rela‐ tive intensity of proteins on day 7 was quantified. Values are means ± SD of triplicate determinations. Data adapted from reference 15.

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in (A). Transfected ODC-VSMCs were then cultured either on plates or in honeycombs for 1, 3, and 7 days. (B) Cell number on days 1 and 3. (C) Western
