Maehara et al. Inflammation and Regeneration (2017) 37:22
DOI 10.1186/s41232-017-0053-6

Inflammation and Regeneration

RESEARCH ARTICLE

Open Access

Characterization of polydactyly-derived
chondrocyte sheets versus adultchondrocyte sheets for articular cartilage
repairMiki Maehara1, Masato Sato1* , Eriko Toyoda1, Takumi Takahashi1, Eri Okada1, Tomomi Kotoku2
and Masahiko Watanabe1

Abstract
Background: We previously conducted a first-in-human clinical study of articular cartilage repair using autologous
chondrocyte sheets and confirmed the regeneration of hyaline-like cartilage in all eight patients. However, regenerative
medicine with autologous chondrocyte sheets requires the harvesting of tissue from healthy regions, and the quality ofthis tissue varies between individuals. To overcome such limitations, allogeneic transplantation is a promising treatment
method, particularly for articular cartilage repair. In this study, we investigated the characteristics of polydactyly-derived
chondrocyte sheets fabricated from the chondrocytes of young polydactyly donors.
Methods: Polydactyly-derived chondrocyte (PD) sheets were fabricated from the tissue obtained from eight polydactyly
donors (average age = 13.4 months). To create these PD sheets, chondrocytes at passage 2 or 3 were seeded on
temperature-responsive culture inserts and cultured for 2 weeks. For comparison, adult chondrocyte sheets
were fabricated from tissue obtained from 11 patients who underwent total knee arthroplasty (TKA; averageage = 74 years). To create these TKA sheets, chondrocytes and synovial cells were cocultured, and the chondrocyte
sheets were triple-layered according to the protocol from our previous clinical study. Cell count, cell viability, cellsurface markers, cell histology, and humoral factors secreted by the sheets were characterized and compared between
the PD sheets and TKA sheets.
Results: Polydactyly-derived chondrocytes proliferated rapidly to establish a layered structure with sufficient
extracellular matrix and formed sheets that could be easily manipulated without tearing. Similar to TKA sheets,
PD sheets expressed aggrecan and fibronectin at the protein level and the surface markers CD44, CD81, andCD90, which are characteristic of mesenchymal cells. PD sheets also produced significantly higher levels oftransforming growth factor beta-1 and lower levels of matrix metalloproteinase-3 than those produced by TKA
sheets, suggesting that young polydactyly-derived chondrocytes have advantages as a potential cell source.
Conclusions: PD sheets exhibited characteristics thought to be important to chondrocyte sheets as well as
proliferative capacity that may facilitate provision of a stable supply in the future.
Keywords: Cartilage regeneration, Cell sheet technology, Chondrocyte sheet, Osteoarthritis, Polydactyly-derived
chondrocyte* Correspondence: sato-m@is.icc.u-tokai.ac.jp
1
Department of Orthopaedic Surgery, Surgical Science, Tokai University
School of Medicine, Kanagawa, Japan
Full list of author information is available at the end of the article© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Maehara et al. Inflammation and Regeneration (2017) 37:22

Background
Articular cartilage is composed mainly of hyaline cartilage, which exhibits viscoelastic properties. Because of its
low cellularity and avascular nature, its capacity to selfregenerate after injury or degeneration is limited [1].
Existing treatment methods such as subchondral drilling[2], microfracture [3, 4], and mosaicplasty [5, 6] are all
symptomatic therapies that typically fill defects with inferior fibrocartilage, which lacks the mechanical properties exhibited by native hyaline cartilage. Since being
first reported by Brittberg et al. [7] in 1994, autologous
chondrocyte implantation (ACI) has been performed
widely as an attempt to regenerate articular cartilage.
However, regeneration with fibrocartilage or with a mixture of fibrocartilage and hyaline cartilage has been reported [8], and the advantage of ACI over existing
methods is controversial [9]. As such, regeneration of
hyaline cartilage is a challenge for regenerative medicine
and is considered important in providing a long-term
treatment.
In regenerative medicine, cell sheet technology [10, 11]
has been applied to the regeneration of various tissues including the cornea [12], esophagus [13], myocardium [14],and periodontal tissue [15]. Culture dishes coated withtemperature-responsive polymers [16, 17] allow the collection of cells as cell sheets without the use of digestive
enzymes. The collection of cells with the extracellularmatrix, cell-to-cell connections, and surface proteinsintact makes this technology suited to regenerative
medicine.
We have applied this technology to articular cartilage
repair by developing chondrocyte sheets (i.e., sheets derived from chondrocytes) for the treatment of cartilage
defects. We previously reported that culturing chondrocytes on temperature-responsive culture inserts andlayering three sheets formed a strong three-dimensional
structure [18]. We subsequently investigated the effectiveness of such layered chondrocyte sheets for the repair offull-thickness defects in rats [19], rabbits [20], and minipigs [21] and for the repair of partial-thickness defects inrabbits [22]. Having produced such evidence, we conducted a clinical study with autologous chondrocyte
sheets and treated eight patients with cartilage defects
accompanied by osteoarthritis. No adverse events weredetected, and improvements in both clinical scores and regeneration of hyaline cartilage were confirmed in all patients (manuscript in preparation).
However, the fabrication and transplantation of autologous chondrocyte sheets require two surgeries, and theproliferative capacity of chondrocytes also varies greatly
between individuals. To overcome these issues, we investigated the possibility of using allogeneic cell sources.
Allogeneic transplantation of chondrocytes is known to
be immunologically tolerated, and particulated juvenile

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cartilage implants (De Novo NT®; Zimmer, Warsaw, IN,
USA) are used clinically in the USA [23]. To ensure
traceability, we focused on the surgical remains obtained
from polydactyly patients at Tokai University Hospital asa source of allogeneic chondrocytes.
In this study, we collected chondrocytes from polydactyly
donors and fabricated polydactyly-derived chondrocyte(PD) sheets on temperature-responsive culture inserts. For
comparison, adult chondrocyte sheets were fabricated fromtissue obtained from patients who underwent total knee
arthroplasty (TKA), hereafter called TKA sheets. To investigate the potential of using PDs as a cell source clinically,
we compared the properties of PD sheets with those of
TKA sheets.

Methods
All experiments were conducted with the approval of
the Tokai University Ethics Committee and with either
informed consent from the patient or parental
permission.
Fabrication of PD sheets

Cartilage tissue was obtained from eight patients (average age 13.4 months, range 8–17 months, four boys andfour girls) who underwent polydactyly surgery at Tokai
University Hospital. A summary of the PD sheet fabrication process is shown in Fig. 1a. Cartilage tissue wasminced with scissors and subsequently incubated in
Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12;
Gibco, Grand Island, NY, USA) supplemented with 20%
fetal bovine serum (FBS; AusGeneX, Molendinar,
Australia), 1% antibiotic–antimycotic solution (AB; Gibco),and 5 mg/mL collagenase type 1 (CLS1; Worthington,
Mannheim, Germany) for 1.5 h at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. The cell suspension waswashed and passed through a 100-μm strainer (BD Falcon,
Franklin Lakes, NJ, USA).
The collected cells were seeded at a density of 1 × 104
cells/cm2 on six-well culture plates (Corning, Corning, NY,
USA) in DMEM/F12 supplemented with 20% FBS and 1%
AB and incubated at 37 °C. After 4 days, 100 μg/mL ascorbic acid (Nissin Pharmaceutical, Yamagata, Japan) was
added to the medium and the medium was replaced every
3 or 4 days. Cells were passaged once or twice when theyreached confluence and then cryopreserved. To fabricate
PD sheets, cells were thawed and passaged once and thenseeded on temperature-responsive culture inserts (CellSeed
Inc., Tokyo, Japan) at 1 × 104 cells/cm2. After 2 weeks, theculture plates were kept at 25 °C for 30 min to promote detachment of PD sheets from the inserts and the sheets
were collected onto a polyvinylidene difluoride (PVDF)
membrane. PD sheets were manipulated and visually confirmed to check for strength and any tearing.

Maehara et al. Inflammation and Regeneration (2017) 37:22

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Fig. 1 Protocol for the fabrication of PD sheets and TKA sheets. a Fabrication of PD sheets. PDs obtained from polydactyly surgery were passaged
once or twice and stocked at − 180 °C. After further expansion, P2 or P3 cells were seeded on temperature-responsive culture inserts and cultured
for 2 weeks. b Fabrication of TKA sheets. Adult chondrocytes and synovial cells obtained from TKA surgery were stocked at P0 and P1, respectively.
Chondrocytes were seeded on temperature-responsive culture inserts and cocultured with synovial cells for 2 weeks. Three chondrocyte sheets werelayered and cultured for an additional week

Fabrication of adult chondrocyte sheets

Adult knee articular cartilage and synovium were obtained from 11 patients (average age 74 years, range 67–
79 years, five men and six women) who underwent TKA
surgery at Tokai University Hospital. TKA sheets were
fabricated according to previously reported methods[24, 25], which are similar to those used to create autologous chondrocyte sheets in our clinical study. A
summary of the TKA sheet fabrication process is shownin Fig. 1b.
Briefly, cartilage and synovium were minced and subsequently incubated in DMEM/F12 supplemented with
20% FBS, 1% AB, and 5 mg/mL CLS1 for 4 and 2 h, respectively, at 37 °C in a humidified atmosphere of 5%
CO2 and 95% air. The cell suspensions were washed andpassed through 100-μm strainers. Chondrocytes were
cryopreserved, and synovial cells were seeded at 1 × 104
cells/cm2 and cryopreserved after confluence. To fabricate
TKA sheets, chondrocytes were seeded on temperatureresponsive culture inserts and cocultured with synovial
cells for 2 weeks and three chondrocyte sheets werelayered onto a PVDF membrane and cultured further for
1 week. TKA sheets were then manipulated and visuallyconfirmed to check for strength and any tearing.

P (Roche, Basel, Switzerland) at 37 °C for up to 30 min
and then centrifuged at 1500 rpm for 5 min. The isolated cells were finally resuspended in DMEM/F12, andcell count and viability were determined using the trypan
blue exclusion assay.
Flow cytometric analysis

After obtaining the cell count, isolated cells were washedwith DPBS containing 0.2% bovine serum albumin (BSA;
Sigma-Aldrich, St. Louis, MO, USA) and 1 mM ethylenediaminetetraacetic acid (EDTA; Gibco). About
1.5 × 105 cells were mixed in each tube with the following antibodies: hCD31–fluorescein isothiocyanate (FITC)(clone: 5.6E, Beckman & Coulter, Brea, CA, USA),hCD44–FITC (clone: G44-26), hCD45–FITC (clone:
J.33, Beckman & Coulter), hCD81–allophycocyanin(APC) (clone: JS-81, BD Bioscience, Franklin, NJ, USA),and hCD90–APC (clone: 5E10, BD Bioscience). The
cells were incubated for 90 min at 4 °C and thenwashed with DPBS containing 0.2% BSA and 1 mM
EDTA. Fluoroprobe-labeled mouse IgG1 antibody(clone: 679.1Mc7, Beckman & Coulter) and mouse
IgG2b antibody (clone: MG2b-57, Beckman & Coulter)
were used as negative controls. Stained cells were analyzed using a FACSVerse™ cell sorter (BD Bioscience).

Cell count and viability

PD sheets and TKA sheets were washed in Dulbecco’s
phosphate-buffered saline (DPBS; Gibco). The sheets
were then incubated in TripLE Express® (Gibco) at 37 °C
for 15 min and centrifuged at 1500 rpm for 5 min. The
cell sheets were resuspended in 0.25 mg/mL Collagenase

Histological and immunohistochemical staining

PD sheets and TKA sheets were harvested after culture
and then embedded and frozen in optimal cutting
temperature compound (Sakura Finetek Japan, Tokyo,
Japan). Then, sections 10 μm thick were stained forMaehara et al. Inflammation and Regeneration (2017) 37:22

proteoglycans with Safranin O or toluidine blue using
standard methods. Sections 20 μm thick were immunostained with anti-human type I collagen (COL1; 1:200;
Southern Biotech, Birmingham, AL, USA), type II collagen(COL2; 1:200; Kyowa Pharma Chemical, Toyama, Japan),fibronectin (FN; 1:500; Merck, Darmstadt, Germany), andaggrecan (ACAN; 1:10; R&D Systems, Minneapolis, MN,
USA) at 4 °C overnight. The sections were washed and incubated at room temperature for 1 h with the secondary
antibody Alexa Fluor 488-conjugated goat anti-mouse Ig(Thermo Fisher Scientific, MA, USA) for COL2 and FN
or Alexa Fluor 546-conjugated donkey anti-goat Ig(Thermo Fisher Scientific) for COL1 and ACAN. After
immunostaining, the sections were washed and mountedwith VECTASHIELD Antifade Mounting Medium with
4′,6-diamidino-2-phenylindole (Vector Laboratories,
Burlingame, CA, USA). Microscopic images were captured
under a BZ-8000 microscope (Keyence, Osaka, Japan).
Measurement of humoral factors

A random selection of fabricated PD sheets and TKA
sheets were cultured for 72 h in 3 mL of DMEM/F12 supplemented with 1% FBS and 1% AB. Supernatants were collected and centrifuged at 15,000g for 10 min to remove celldebris. The concentrations of transforming growth factor
beta-1 (TGF-β1; R&D Systems), melanoma inhibitory
activity (MIA; Roche), tissue inhibitor of metalloproteinases(TIMP1; R&D Systems), matrix metalloproteinase-3(MMP3; Sigma-Aldrich), stanniocalcin-1 (STC1; Cusabio,
College Park, MD, USA), and hyaluronan and proteoglycan
link protein 1 (HAPLN1; US Biological, Salem, MA, USA)
were measured using enzyme-linked immunosorbent assay(ELISA) kits. The signal detected for blank medium containing 1% FBS was subtracted to adjust for proteins contained in FBS. Measurements were repeated at least twice
for each donor, and averages were used.
Statistical analysis

Numerical results are expressed as mean and standard
deviation. Statistical analysis was performed using SPSS
23.0 software (IBM Corp., Armonk, NY, USA). Differences
between the two groups were identified using Student’s ttest. The level of significance was set at P < 0.05.

Page 4 of 10

proliferated 7.8 times and P2 cells proliferated 4.8 times.
From the polydactyly tissue obtained from one donor,
we calculated that, theoretically, 693 P2 sheets and 3326
P3 sheets can be fabricated (Table 1). The average wetweight of cartilage tissue obtained from TKA donors
was 9.2 g, and the number of cells collected after enzymatic digestion was 18.5 × 106 cells. However, theoretically, only 29 layered TKA sheets can be created from theP0 cells (Table 1). Furthermore, only 1 to 3 g of cartilage
tissue was collected during the clinical trial; therefore,
the number of autologous chondrocyte sheets that were
fabricated in the clinical trial was three to seven sheets.
Cell count, sheet thickness, and macroscopic observations

Regardless of the type of donor, all PD sheets were a highdensity of cells through the 2 weeks of culture and withoutlayering (Fig. 2d). PD sheets and TKA sheets were easily
harvested and manipulated without tearing (Fig. 3a, b). An
average PD sheet contained 2.6 ± 0.8 × 106 cells with anaverage thickness of 15.2 ± 4.0 μm. After a total of 3 weeks
of culture, TKA sheets formed a thick structure with integrated layers (Fig. 3h). An average TKA sheet contained 1.6 ± 0.1 × 106 cells with an average thicknessof 45.5 ± 14.6 μm.
Histological and immunohistochemical analyses

Histological evaluation showed that, for all donors, both
PD sheets and TKA sheets stained weakly or did notstain for Safranin O or toluidine blue (Fig. 3b, c, i, j). Immunohistochemical analysis showed negative staining
for COL2 and positive staining for COL1, FN, and
ACAN (Fig. 3d–g, k–n).
Flow cytometric analysis

PD sheets and TKA sheets showed similar surface
markers (Fig. 4). Both PD sheets and TKA sheets were
negative for CD31 and CD45 (PD sheets: CD31 = 0.1%,
CD45 = 0.1%; TKA sheets: CD31 = 0.0%, CD45 = 0.0%).
Both PD sheets and TKA sheets were positive for CD44,
CD81, and CD90 (PD sheets: CD44 = 99.8%, CD81 = 99.9%,
CD90 = 99.8%; TKA sheets: CD44 = 98.7%, CD81 = 98.5%,
CD90 = 99.0%).
Measurement of humoral factors

Results
Isolation and proliferation of PDs

The average wet weight of cartilage tissue obtained frompolydactyly donors was 0.3 g, and the average number ofcells collected after enzymatic digestion was 0.17 × 106
cells (Table 1). Cultured cells proliferated rapidly andreached confluence around day 5 (Fig. 2b), and cells
grew to an average density of 3.7 × 106 cells or 22 times.
Passage 1 (P1) or passage 2 (P2) cells were collected andstocked at − 180 °C. After further expansion, P1 cells

The concentrations of humoral factors secreted by PD
sheets and TKA sheets are summarized in Fig. 5. PD
sheets produced higher concentrations of TGF-β1 (PDsheets 1.96 to 3.21 ng/mL; TKA sheets 0.55 to2.58 ng/mL), MIA (PD sheets 9.88 to 35.15 ng/mL;
TKA sheets 8.76 to 26.45 ng/mL), and TIMP1 (PDsheets 582.60 to 979.60 ng/mL; TKA sheets 83.71 to
798.10 ng/mL). TKA sheets produced higher concentrations of MMP3 (PD sheets 5.26 to 22.83 ng/mL;
TKA sheets 37.29 to 84.90 ng/mL), STC1 (PD sheets 89.00

Maehara et al. Inflammation and Regeneration (2017) 37:22

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Table 1 Theoretical number of chondrocyte sheets that can be fabricated from the collected cartilage tissue
Wet weight of harvested Cell count after enzymatic Passage of cells used Seeding cell density on Number ofto fabricate sheets
temperature-responsive layers
cartilage tissue (g)digestion (× 106)
culture inserts
PD sheets

0.3 ± 0.2

0.17 ± 0.09

TKA sheets 9.2 ± 1.0

18.5 ± 6.52

1 × 104 cells/cm2

1

5 × 104 cells/cm2

3

30

Theoretical numberof sheets that canbe fabricated
693
3326
29

PD polydactyly-derived chondrocyte, TKA total knee arthroplasty

to 186.60 ng/mL; TKA sheets 153.20 to 626.60 ng/mL),and HAPLN1 (PD sheets 30.05 to 33.21 ng/mL; TKA
sheets 37.59 to 43.98 ng/mL). The concentrations differedsignificantly between PD sheets and TKA sheets for TGFβ1, MMP3, and HAPLN1.

Discussion
The objectives of this study were to characterize sheets
fabricated from polydactyly-derived chondrocytes and tocompare their properties with those of TKA sheets fabricated from adult chondrocytes. For autologous transplantation, the amount of cartilage tissue that can becollected from nonloading regions is limited, and because of the adult chondrocyte’s limited proliferative
capacity, coculture with synovial cells to improve proliferation and layering of chondrocyte sheets to increase
the secretion of cartilage anabolic factors [24, 26] is necessary. Our results suggest that PDs can proliferate
rapidly without coculture. Theoretically, more than 600
PD sheets can be fabricated from P2 cells and more than3000 PD sheets can be fabricated from P3 cells, whereas
the number of autologous chondrocyte sheets that canbe fabricated is limited. The possibility of providing astable supply from a single lot is also attractive for ensuring the quality and safety of chondrocyte sheets.
PD sheets were found to secrete a sufficient amount of
extracellular matrix and formed a layered structure during
culture without having to be physically layered. This property gives an added advantage during the fabrication
process of PD sheets; that is, a single layer of PD sheets, although thinner than layered TKA sheets, displayed enoughstrength to resist tearing and to be manipulated for transplantation. PD sheets strongly expressed ACAN and FN
and were negative for CD31 and CD45, which confirmedthat there was no contamination with hematopoietic cells.
Similar to TKA sheets, PD sheets also expressed mesenchymal surface markers CD44, CD81, and CD90 and secreted
various humoral factors related to cartilage anabolism.
Histological and immunohistochemical analyses revealed that PD sheets and TKA sheets did not stain for
Safranin O, toluidine blue, or COL2. However, the autologous chondrocyte sheets that promoted hyaline cartilage repair used in our clinical study also did not stain
for Safranin O, toluidine blue, or COL2 (manuscript in

Fig. 2 Microscopic view of the proliferation of PDs. PDs seeded on temperature-responsive culture inserts were observed on day 3 (a), day 5 (b),day 7 (c), and day 14 (d) (×100). Cells reached confluence at day 5 and, by day 14, they were tightly packed. (Scale bar = 100 μm)

Maehara et al. Inflammation and Regeneration (2017) 37:22

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Fig. 3 Macroscopic images and microscopic images of histological sections. Images of PD sheets (a–g) and TKA sheets (h–n). a, h Representative
images of a PD sheet and TKA sheet attached to a white PVDF membrane. Histological staining for Safranin O (b, i) and toluidine blue (c, j) showedeither weak or no staining for all donors (×20). Immunostaining for COL1 (red; d, k), COL2 (green; e, l), ACAN (red; f, m), and FN (green; g, n) showed
positive staining for COL1, ACAN, and FN but negative staining for COL2 (×20). (Scale bar = 100 μm)

preparation), suggesting that chondrocyte sheets may behave differently in vivo.
The regenerative effects of chondrocyte sheets may be
attributed to the protection of cartilage defects fromcatabolic factors within the synovial fluid, prevention ofproteoglycan loss, and continued secretion of cartilage
anabolic factors by transplanted chondrocytes that act asinitiators of cartilage repair through the recruitment ofstem cells from the bone marrow. PD sheets were foundto secrete TIMP1, STC1, and HAPLN1, in addition to
TGF-β1 and MIA as previously reported for TKA sheets[26]. TIMP1 inhibits the catabolic activity of MMP1 and
MMP3 [27], and STC1 is involved in the regulation ofangiogenesis [28] and inhibition of cartilage hypertrophyand bone formation of growth plate cartilage [29].
HAPLN1 stabilizes the association of ACAN andhyaluronan [30]. Compared with TKA sheets, PD sheets
secreted significantly less MMP3, a known catabolic factor [31]. We also detected donor differences in PD
sheets for humoral factor concentrations (Fig. 5), indicating that selection of donors based on the secretionlevels of humoral factors may be necessary. One limitation of our study was that TKA sheets were fabricatedfrom patients whose ages ranged from 67 to 79 years,
and cartilage tissue was collected in bulk from areas thatappeared normal. The autologous chondrocyte sheets
used in our clinical study were fabricated from patients
whose ages ranged from 30 to 59 years, and cartilage tissue was collected from the nonloading regions of knee
cartilage. Although TKA sheets were shown to be effective in a rabbit xenogeneic transplantation model (manuscript in preparation), comparisons must be made

Maehara et al. Inflammation and Regeneration (2017) 37:22

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Fig. 4 Flow cytometric analysis of surface markers. Results are shown for the flow cytometric analysis of hematopoietic cell markers CD31 andCD45 and mesenchymal stem cell markers CD44, CD81, and CD90. The target markers are indicated in red, and IgG isotype controls are indicatedin gray. Both PD sheets and TKA sheets were negative for CD31 and CD45 and positive for CD44, CD81, and CD90

Maehara et al. Inflammation and Regeneration (2017) 37:22

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Fig. 5 A combination of box and strip plot of the concentrations of humoral factors secreted by PD sheets and TKA sheets. Each circle indicatesthe average value for a single donor. Upper and lower boxes represent the 25 and 75 percentiles, lines inside the boxes are medians, upper andlower whiskers represent 1.5 times the interquartile range, and circles outside of whiskers represent outliers. PD sheets produced significantly higher
concentrations of TGF-β1, and TKA sheets produced significantly higher concentrations of MMP3 and HAPLN1. *P < 0.05

carefully because TKA sheets may be inferior to those
sheets used in our clinical study.
Here, we fabricated and evaluated PD sheets from P2 andP3. Current data suggest that the properties of P2 and P3
PD sheets are not significantly different (data not shown).
However, the loss of cartilage properties through the passaging of PDs has been reported [32] and must be investigatedfurther. Moreover, the in vivo characteristics of PD sheets
are still under investigation. We are currently performing
xenogeneic transplantation of human PD sheets into rat andrabbit osteochondral defect models to evaluate their in vivo
efficacy. In addition, to make this treatment widely available
off the shelf, we are investigating vitrification methods anddeveloping systems for storing PD sheets [33, 34].
We found that PD sheets and adult TKA sheets shareimportant characteristics, and further investigation of

their in vivo efficacy will help to provide the evidence
necessary to establish PDs as a cell source for allogeneic
chondrocyte sheets.

Conclusions
In this study, we characterized sheets created from PDs
and compared key properties with those of TKA sheets.
PDs proliferated rapidly to establish a layered structurewith sufficient extracellular matrix and formed sheets that
could be easily manipulated without tearing. Similar to
TKA sheets, PD sheets expressed ACAN and FN at theprotein level and produced significantly higher levels of
TGF-β1 and lower levels of MMP3 than those produced
by TKA sheets, suggesting their potential in future clinicalapplications.

Maehara et al. Inflammation and Regeneration (2017) 37:22

Abbreviations
AB: Antibiotic–antimycotic solution; ACAN: Aggrecan; BSA: Bovine serum albumin;
CLS1: Collagenase type 1; COL1: Type I collagen; COL2: Type II collagen;
DPBS: Dulbecco’s phosphate-buffered saline; EDTA: Ethylenediaminetetraacetic
acid; ELISA: Enzyme-linked immunosorbent assay; FBS: Fetal bovine serum;
FN: Fibronectin; HAPLN1: Hyaluronan and proteoglycan link protein 1;
MIA: Melanoma inhibitory activity; MMP3: Matrix metalloproteinase-3; P1: Passage
1; P2: Passage 2; PD: Polydactyly-derived chondrocyte; PVDF: Polyvinylidene
difluoride; STC1: Stanniocalcin-1; TGF-β1: Transforming growth factor beta-1;
TIMP1: Tissue inhibitor of metalloproteinases; TKA: Total knee arthroplasty
Acknowledgements
We thank Professor Teruo Okano and Professor Masayuki Yamato (Institute ofAdvanced Biomedical Engineering and Science, Tokyo Women’s Medical
University) for expert advice on developing cell sheets.
Funding
This research was supported by the Research Project for Practical Applicationsof Regenerative Medicine (No. 12103253 to MS) from the Japan Agency for
Medical Research and Development.
Availability of data and materials
The datasets used and/or analyzed during the current study are available
from the corresponding author on reasonable request.
Authors’ contributions
MS, MW, and ET conceived and designed the study. MM performed the cell
culture, experiments, and statistical analysis, prepared the tables and figures,and wrote the manuscript. ET and TT wrote the manuscript together with
MM. EO and TK performed the cell culture and experiments together with
MM. MS and MW supervised the experiments and provided advice for MM.
All authors read and approved the final manuscript.
Ethics approval and consent to participate
All experiments were conducted with the approval of the Tokai University
Ethics Committee and with either informed consent from the patient orparental permission.
Consent for publication
Not applicable.
Competing interests
MS receives research funding from CellSeed Inc.

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Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations.

22.

Author details
1
Department of Orthopaedic Surgery, Surgical Science, Tokai University
School of Medicine, Kanagawa, Japan. 2CellSeed Inc., Tokyo, Japan.

23.

Received: 21 August 2017 Accepted: 26 September 201724.
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