Effect of poly(3-hydroxyalkanoates) as natural polymers on mesenchymal stem cells

Table 1 The effect of natural poly(3-hydroxyalkanoates) on the growth and differentiation of stem cells

The type of PHA	Surface topography/modification, 3D-scaffolds microstructure	The type of stem cells	The time of cell cultivation	The effect of PHAs on the proliferation of stem cells	The effect of PHAs on the morphology of stem cells	The effect of PHAs on the differentiation of stem cells	Ref.
Osteogenic differentiation
PHB (Mw = 150000) doped with PEG 1000	Porous flat and cubic scaffolds produced by salt leaching from polymer solution using sucrose crystals and ammonium carbonate as porogens	Rat bone marrow MSCs	1, 3, 6, 14 d (proliferation tests); 6, 14 d (differentiation tests)	Suppressed cell proliferation in comparison with TCPS (XTT assay)	Good quality of cell adhesion and spread with developed filopodia (SEM)	Spontaneous osteogenic differentiation in regular medium (increased ALP activity on the 7th and 14th days by up to 10 times and CD45 expression in comparison with TCPS) (ALP activity assay, immunocytochemistry using flow cytometer)	[14,28]
PHBV (with 5 mol % 3-HV)	Porous flat scaffolds produced by salt leaching from polymer solution using sucrose crystals as porogens; oxygen plasma treated and untreated	Rat bone marrow stromal osteoblastic cells (rat bone marrow MSCs)	7, 14, 21, 28 d	Low cell proliferation (60% at day 7 for untreated scaffolds). Treatment with oxygen plasma slightly increased (up to 50%) cell growth (MTS)	-	Spontaneous osteogenic differentiation in regular medium (increased ALP activity on the 28th day by up to 10 times). Treatment with oxygen plasma did not change this effect (ALP activity assay)	[22]
PHBV (with 8 mol % 3-HV)	Porous flat scaffolds produced by salt leaching from polymer solution using sucrose crystals as porogens; oxygen plasma treated and untreated	Rat bone marrow stromal osteoblastic cells (rat bone marrow MSCs)	7, 14, 21, 29, 60 d	Low cell proliferation (2.2 times at day 60 for untreated scaffolds). Treatment with oxygen plasma slightly increased (up to 23%) cell growth (MTS)	Spindle shaped cells on the 29th day with cytoplasmic extensions; large ovoid cells with osteoblast-like morphology on the 60th day. Mineralization from day 21 to day 60. A close connection between the cell boundary and the scaffold (SEM, histology, CM)	Spontaneous osteogenic differentiation in regular medium (increased ALP activity and osteocalcin expression at day 60 by up to 12 times and 4 times, respectively). Treatment with oxygen plasma did not change the induction of ALP activity or increase the induction of osteocalcin expression (ALP activity assay, test for osteocalcin)	[17]
PHBV	Porous scaffolds produced by freeze-drying	Human adipose-derived MSCs	2, 7, 14, 21, 28 d	Increased cell proliferation from days 7 to 28 (MTT)	Good quality of cell adhesion and spread with developed cytoplasmic extensions (SEM)	Signs of spontaneous osteogenic differentiation in regular medium (slightly increased ALP activity at day 28 by up to 10 times). Inhibition of osteogenic differentiation (ALP activity assay) or not (proteins expression assays) in osteogenic medium (osteopontin, collagen I type, osteocalcin indirect immunofluorescence)	[12]
PHB	Porous cubic scaffolds produced by salt leaching from polymer solution using sucrose crystals as porogens	Rat bone marrow and adipose-derived MSCs	3, 7, 14, 21 d (proliferation tests); 7, 14, 21 d (differentiation tests)	Low cell proliferation (up to 2 times at day 21) (MTT assay)	Spherically shaped cells grouped within cell clusters at day 21; calcium deposition (SEM)	Signs of spontaneous osteogenic differentiation in regular medium (a very slight increase in ALP activity and osteocalcin expression at day 21 by up to 4 times); osteocalcin expression; and calcium deposition at day 21 (ALP activity assay, von Kossa staining, PCR for osteocalcin).	[15]
PHBV (with 12 mol % 3-HV) doped with poly(ethylene oxide) (Mw ≈ 1000000) with a mass ratio of 9:1	Scaffolds produced by electrospinning with randomly oriented nanofibers	Rat bone marrow MSCs	1, 4, 7 d (proliferation tests); 7, 14 d (differentiation tests)	Relatively high cell proliferation rate (up to 3-fold at day 7 (CCK-8 test)	Good quality of cell spread in random directions, filopodia extend along the fibers (SEM)	Slight signs of spontaneous osteogenic differentiation in regular medium (a very slight increase in ALP expression at day 14) (ALP staining, PCR), or no effect (staining and expression of osteocalcin, calcium deposition)	[16,21]
PHB, PHBHHx (Mw = 470000)	Porous flat scaffolds produced by salt leaching from polymer solution using salt crystals as porogens	Rabbit bone marrow MSCs	10 d	Higher proliferation in comparison with PLA (MTT assay)	Typical osteoblast phenotype, calcium deposition at day 10 (SEM)	Spontaneous osteogenic differentiation in regular medium at day 10 (ALP activity assay)	[18]
PHBV (with 5 mol % 3-HV)	Films casted from polymer solution	Murine calvarial preosteoblast cells MC3T3-E1 S14 line	21 d	-	A dense monolayer of cuboidal-shape cells with obvious areas of mineralization (CM)	Higher stimulation of osteogenic differentiation in comparison with cells grown on TCPS in osteogenic medium (Calcium C test, von Kossa staining)	[19]
PHBV (with 12 mol % 3-HV; Mw = 530000)	Scaffolds produced by electrospinning with randomly oriented fibers	Human bone marrow MSCs	1, 7, 11 d (proliferation tests); 7 d (differentiation tests)	No difference between cell proliferation on scaffolds from PHBV and poly-ε-caprolactone (trypan blue assay)	Good quality of cell adhesion and spread with developed filopodia. Cells have a nearly spherical shape (CM)	Higher modulation of osteogenic differentiation in comparison with MSCs grown on poly-ε-caprolactone scaffolds in osteogenic medium (calcein and Alizarin red staining assay)	[13]
PHBV (with 5 mol % 3-HV, Mw = 680000)	Scaffolds produced by electrospinning with randomly oriented nanofibers	Human induced pluripotent stem cells	1, 3, 5, 7, 10 d (proliferation tests); 7, 14 d (differentiation tests)	The higher proliferation rate in comparison with TCPS (MTT assay)	Original-like MSCs formed colonies at day 5 (LM).	Higher stimulation of osteogenic differentiation in comparison with cells grown on TCPS in osteogenic medium (ALP activity assay, RT-PCR for measure of ranx-2, col-1, ALP, osteonectin, osteocalcin expression levels; Western blot assay for osteocalcin and osteopontin expression levels)	[20]
PHBHHx (with 12 mol % 3-HHx), and PHBVHHx	Films casted from polymer solution	Human bone marrow MSCs	4, 72 h (proliferation tests); 14, 21 d (differentiation tests)	Higher cell proliferation rate (for PHBHHx and PHBVHHx) in comparison with PLA and with TCPS (for PHBVHHx)	-	No effect on cell differentiation in regular medium (FM: ALP and van Kossa staining)	[23]
PHBHHx (with 8.3 mol % 3-HHx, Mw = 1210000)	Smooth compression-molded films, porous scaffolds casted from solution films, electrospun scaffolds	Human bone marrow MSCs	5, 6, 7, 14 d (proliferation tests); 14 d, 5 wk (differentiation tests)	The same (for compression-molded films) and higher (up to 2-fold for solution-casted and electrospun films) cell proliferation in comparison with TCPS	Spindle-like, similar to original MSCs, good cell adhesion and spreading in regular medium (FM, SEM).	No differentiation in regular medium. Inhibition of differentiation in osteogenic medium (osteocalcin assay, Alizarin red S staining, RT-PCR for measure of collagen I and osteonectin expression level)	[24]
PHBHHx (Mw = 300000)	Scaffolds produced by electrospinning with randomly oriented fibers	Rat bone marrow MSCs	3 d	-	The well-developed stress fibers spanned the entire cell body and supermature focal adhesions (CM, immunofluorescence)	No osteogenic differentiation: no significant expression of osteocalcin, osteopontin, osteonectin, runx2, in regular and osteogenic media at day 3 (RT-PCR)	[25]
Chondrogenic differentiation
PHB and PHBHHx in a ratio of 1:2 (by weight)	Porous flat scaffolds with pores of 200–300 μm in diameter produced by salt leaching from polymer solution using salt crystals as porogens and lyophilization	Human adipose-derived stromal cells	7 d (proliferation tests); 14 d, 5 wk (differentiation tests)	100% cells viability at day 7 (FM)	After 1 d, the differentiated cells attached to scaffolds. At 7 d and 14 d, the differentiated cells produced extracellular matrices to fill the voids of the scaffolds (SEM)	Chondrogenic differentiation in chondrogenic medium: increased glycosaminoglycan and collagen content. No chondrogenic differentiation in regular medium: a very slight increase in collagen content (biochemical glycosaminoglycan and collagen content assays)	[27]
PHBHHx (Mw = 600000)	Films casted from polymer solution	Murine bone marrow MSCs; chondrocytes isolated from cartilage from knees of mice	1 d (24 h)	No change in cell proliferation in comparison with TCPS (RT-PCR analysis of proliferating cell nuclear antigen)	-	Spontaneous chondrogenic differentiation in regular medium at day 1 (as in chondrocytes with the exception of col1): upregulation of aggrecan, col2, sox9, col10, pthrp, and col1 genes, downregulation of osteocalcin, Cbfa1/Runx2, MMP13 genes, microRNAs miR-29a and miR-29b (alcian blue staining for glycosaminoglycans; immunostaining for type II collagen; RT-PCR analysis of chondrogenic markers)	[26]
PHBV	Porous cylindrical (5 mm diameter, 2 mm thick) scaffolds with a pore size of 30-300 μm produced by salt leaching	Swine bone marrow MSCs, cartilage progenitor cells, and chondrocytes	After 1 wk of in vitro culture subcutaneous implantation in vivo into nude mice for 6 wk.	Higher wet weight and volume of the cell-scaffold constructs seeded with cartilage progenitor cells and chondrocytes in comparison with MSCs 6 wk after implantation.	Good compatibility between the cells and the scaffold and production of considerable amounts of extracellular matrix after 1 wk of in vitro culture (SEM)	Higher chondrogenic differentiation in vivo of cartilage progenitor cells and chondrocytes: increased expression of sox-9, collagen II, aggrecan, safranin-O, glycosaminoglycans in comparison with MSCs (alcian blue staining, immunostaining, RT-PCR)	[29]
PHBHHx	Films casted from polymer solution	Human umbilical cord MSCs	3, 7, 14 d (proliferation tests); 14, 28 d (differentiation tests)	Higher MSCs proliferation in chondrogenic medium in comparison with TCPS (MTT assay)	Good spreading of cells and their proliferation (SEM)	Chondrogenic differentiation in chondrogenic medium: increased glycosaminoglycan, proteoglycan, and collagen content, upregulation of collagen II and aggrecan, genes (amino-sugars and hydroxyproline assays, toluidine blue staining, RT-PCR)	[28]
Epidermal differentiation
PHBV, (Mw = 450 kDa)	Scaffolds produced by electrospinning with randomly oriented nanofibers	Bone marrow MSCs	1, 3, 7, 14 d (proliferation tests); 3, 7, 14 d (differentiation tests)	100% viability of cells. Higher cell proliferation in epidermal-induction medium in comparison with regular medium. Lower cell proliferation on PHBV scaffolds in regular and epidermal-induced medium in comparison with TCPS (CM, MTS assay)	Good adhesion and spreading of cells tended to start forming a monolayer at day 7. The spindle-like, fully extended morphology of MSCs at day 3. The keratinocytic morphology of MSCs characterized by polygonal cells at days 7 and 14 (SEM, CM)	The epidermogenic differentiation in epidermal-induction medium: increased expression of keratin, filaggrin, and involucrin. No epidermogenic differentiation in regular medium (CM, RT-PCR)	[38]
Adipogenic differentiation
PHBHHx (Mw = 300000)	Scaffolds produced by electrospinning with randomly oriented fibers	Rat bone marrow MSCs	3 d	-	Well-developed stress fibers spanned the entire cell body and supermature focal adhesions (CM, immunofluorescence)	No adipogenic differentiation: no significant expression of PPARg, Lpl, ADFP, CD36 in regular and adipogenic media at day 3 (RT-PCR)	[25]
Endotheliogenic differentiation
PHB/PHBV composite (30:70)	Scaffolds produced by electrospinning with randomly oriented nanofibers	Human adipose tissue-derived MSCs	7, 14, 21 d (proliferation tests); 7, 14, 21 d (differentiation tests)	Lower cell proliferation on scaffolds in endothelial-induction medium in comparison with TCPS. Higher cell proliferation on scaffolds in regular medium in comparison with TCPS (CM, MTT assay)	Good adhesion and spread, typical spindle-shape morphology, and cell-to-cell interactions. Good distribution of cells in regular medium. MSCs in endothelial-induction medium formed circle-like structures characteristic of endothelial cell organization, mimicking the tubular organization of blood vessels at day 21 (SEM, CM, calcein-AM staining)	Endotheliogenic differentiation in endothelial-induction medium: increased expression of VE-Cadherin, vWF factor, and VEGFR2 (immunostaining, flow cytometry, RT-PCR)	[39]
Neurogenic differentiation
PHBHHx and PHBVHHx	Films casted from polymer solution, porous scaffolds with a pore size of 110-170 μm produced by in solution phase separation	Human bone marrow MSCs	2, 3 d (proliferation tests); 7, 14, 21 d (differentiation tests)	Slightly higher cell proliferation on PHBHHx and PHBVHHx films (33% and 31%, respectively) in comparison with PLA films in regular medium at day 3 (CCK-8 assay)	Good adhesion, spread, and proliferation in PHBHHx and PHBVHHx films and PHBVHHx scaffolds in regular medium (SEM, CM)	Neurogenic differentiation of neural stem cells in neurogenic medium: increased expression of nestin, β-tubulin III and anti-glial fibrillary acidic protein (slightly higher expression levels of these markers in cells grown on PHBHHx and PHBVHHx films in comparison with PLA)	[34]
PHBHHx and PHBVHHx	Films casted from polymer solution	Rat neural embryonic stem cells	1, 3, 5 d (proliferation tests); 3, 7 d (differentiation tests)	Same cell proliferation and viability on PHBHHx and PHBVHHx films in comparison with PLA films in neurogenic medium (CCK-8 assay)	Bipolar or even monopolar morphology of the cells with relatively short neuritis at day 3 in neurogenic medium (CM)	Neurogenic differentiation of neural stem cells in neurogenic medium: increased expression of β-tubulin III and anti-glial fibrillary acidic protein (the same level of this marker expression in cells grown on PLA, PHBHHx and PHBVHHx films)	[36]
PHB, PHBV, P3HB4HB, and PHBHHx	Films casted from polymer solution, porous scaffolds produced by in solution phase separation and freeze-drying	Rat neural embryonic stem cells	7, 10 d (proliferation tests); 7, 14, 21 d (differentiation tests)	Slightly higher cell proliferation on PHB, PHBHHx and PHB4HB films in comparison with PLA films in neurogenic medium (CCK-8 assay)	Cells with extended processes and plausible neurite connections at days 3 and 7 in neurogenic medium (CM, SEM)	Neurogenic differentiation of neural stem cells in neurogenic medium: increased expression of β-tubulin III (higher expression level of this marker in cells grown on PHB4HB and PHBHHx in comparison with PHB)	[37]

SEM: Scanning electron microscopy; CM: Confocal microscopy; FM: Fluorescence microscopy; LM: Light microscopy; RT-PCR: Reverse transcription polymerase chain reaction; TCPS: Tissue culture plastic; ALP: Alkaline phosphatase; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MTS: 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; XTT: 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide; PHB: Poly(3-hydroxybutyrate); PHBV: Poly(3-hydroxybutyrate-co-3-hydroxyvalerate); PHBHHx: Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate); MSCs: Mesenchymal stem cells; PHAs: Poly(3-hydroxyalkanoates); PLA: polylactic acid.