Keywords
rotator cuff - suture anchors - bone marrow stem cells - biocompatibility - culture
Introduction
The deeper knowledge achieved in the context of rotator cuff injuries and repair,
together with the progressive optimization of the arthroscopic techniques, has made
the arthroscopic rotator cuff repair a common and effective surgical procedure. Indeed,
evidences of satisfactory outcomes of procedure are well reported in the literature,[1] albeit a high recurrence rate is still described.[2]
Several factors, such as surgical technique, tissue quality, comorbidities and type
of devices, have been reported to affect the outcome of arthroscopic rotator cuff
repair.[3] In particular, a growing interest has been recently focused on the biomechanical
properties of different types of suture anchors used for rotator cuff repair concerning
their stress resistance and suture configurations.[4]
[5]
The different types of anchors have specific advantages and disadvantages, resulting
from their characteristics. The first anchors used in arthroscopic rotator cuff repair
were metallic. They gave an excellent fixation but failure of the suture or migration
of the anchor could lead to cartilage damage.[6] Subsequently, biodegradable anchors were introduced.[7] The first generation of biodegradable anchors had the advantage of combining the
resistance to pullout of the metal anchors with a reduced signal alteration at the
magnetic resonance imaging (MRI). However, complications were reported with the use
of this type of suture anchors, such as suture failure due to early degradation, reactive
synovitis, cyst formation, and osteolysis.[8]
[9]
[10]
[11] Moreover, standard radiographs do not allow for the visualization of a mobilized
biodegradable anchor, and MRI would be needed.[12]
The development of these devices continued to move forward and new anchors were recently
introduced by using new materials. Nevertheless, little consideration has been given
to the possible interaction between these fixation devices and the biological environment.
Indeed, a low biocompatibility could represent a trigger for inflammation or bone
resorption, possibly causing failure of the procedure.
The purpose of this study was to verify the in vitro biocompatibility of three different
types (all-suture anchor, metal anchor, and polyetheretherketone [PEEK] anchor). The
hypothesis of the study was that suture anchors can differ according to the material
in terms of cytotoxicity, immunogenicity, and inhibitory action on osteogenic differentiation.
Methods
Bone Marrow Stem Cells Isolation and Culture
Human bone marrow stem cells (BMSCs) were collected from waste surgical samples at
our institute under written consent of the patients (M-SPER-014.ver7 for the use of
surgical waste). Cells were obtained from the femoral canal of one donor after hip
surgical replacement. The aspirate was washed with phosphate-buffered saline (PBS)
and centrifuged at 623 g for 10 minutes. The pellet containing the mononuclear cells fraction was resuspended
in complete medium (CM), composed of Dulbecco's Modified Eagle Medium (DMEM) High
Glucose (Sigma-Aldrich, St. Louis, Missouri, United States), 10% fetal bovine serum
(FBS, Sigma-Aldrich), 50 U/mL penicillin, 50 mg/mL streptomycin, 2 mM L-glutamine
(Life Technologies, Carlsbad, California, United States) and plated in culture flasks
at a density of 5 × 103 cells/cm2. Cells were maintained in culture and expanded until passage 4.
BMSCs were incubated with three different types of suture anchors: an all-suture anchor
(JuggerKnot; Biomet, Warsaw, Indiana, United States), a metal anchor (ThRevo-FT; ConMed,
Largo, Florida, United States), and a PEEK anchor (Cross-FT; ConMed). BMSC cultures
without the devices were used as control samples. Cells were plated onto 12-well plates
at a seeding density of 10,000 cells/cm2. Culture medium was the same for all specimens: a complete culture medium consisting
of DMEM (Sigma-Aldrich), 10% FBS (GE Healthcare, Chicago, Illinois, United States),
and 1% penicillin–streptomycin–glutamine (Life Technologies).
Cell Viability
Cell viability was assessed after 7 days of culture, by MTT colorimetric assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide; Sigma-Aldrich). According to the protocol, specimens were incubated with
MTT solution at a concentration of 0.5 mg/mL for 4 hours at a temperature of 37°C.
Following solubilization with 100% dimethyl sulfoxide (Sigma-Aldrich), absorbance
was measured by spectrophotometry at 570 nm wavelength.
Osteogenic Differentiation
At passage 4, BMSCs were seeded at the density of 104 cells/cm2 in a 12-well plate. Osteogenic stimulus was provided by culturing cells in CM supplemented
with dexamethasone (10 nM), glycerol 2-phosphate (10 mM), L-ascorbic acid 2-phosphate
(150 µM), and cholecalciferol (10 nM). Cultures with each device were performed in
duplicates and BMSCs without devices were considered as controls. Alkaline phosphatase
(ALP) activity was assessed after 14 days of culture, by enzymatic assay. Briefly,
specimens were lysed in 0.1% Triton X-100 and incubated at 37°C with 1 mM para-nitrophenyl
phosphate in phosphatase buffer (100 mM diethanolamine and 0.5 mM MgCl2). The amount of chromogenic para-nitrophenol (pNP) product was assessed by spectrophotometer.
Readings were taken at 405 nm wavelength, and ALP activity was normalized against
the total protein content determined by bicinchoninic acid (BCA) protein assay (BCA
Protein Assay Kit; Thermo Fisher Scientific, Waltham, Massachusetts, United States).
Calcified matrix deposition was assessed after 21 days of culture, by alizarin red
staining (ARS). Cells were stained with 40 mM Alizarin Red S for 15 minutes. Calcified
matrix-bound dye was extracted by incubation with cetylpyridinium chloride (CPC) in
phosphate buffer (0.1 M) to quantify the amount of calcified matrix. Spectrophotometer
readings were taken at 550 nm wavelength.
Peripheral Blood Leucocytes Isolation and Culture
Human peripheral blood leucocytes (PBLs) were isolated from 10 mL of peripheral blood
from one healthy donor, under informed consent. Briefly, blood was diluted 1:1 with
PBS layered over 10 mL Ficoll Isopaque (GE Healthcare) in a 50-mL tube and centrifuged
for 40 minutes at 400 g. After centrifugation, the nucleated cells at the interface between Ficoll and supernatant
were collected, washed two times with PBS, counted, and then maintained in RPMI medium
with 10% FBS. Cells were then cultured in the presence or absence of the three different
devices for 5 days.
Data Analysis
Data from cell count, U/µg of ALP activity, absorbance at 570 and 550 nm for cell
viability and ARS staining, respectively, were expressed as means ± standard deviation.
Statistical analyses were performed with a statistical software (GraphPad Prism v5.00;
GraphPad Software, La Jolla, California, United States) using the analysis of variance
one-way test with Bonferroni's post hoc test for the evaluation of differences between
single datasets. Level of significance was set at p < 0.05.
Results
Cell Viability
Cell viability of BMSCs, incubated or not with the medical devices, was assessed after
7 days of culture by MTT assay. If compared with the control samples (without devices),
significantly lower viability was observed for BMSCs incubated with ThRevo-FT (−22%;
p < 0.05) and Cross-FT (−28%; p < 0.05). However, no differences were observed among cultures incubated with the
devices ([Fig. 1]).
Fig. 1 Viability assessment of BMSCs after 7 days of culture with or without the suture
anchors (data are expressed as means ± SD). Control: *p < 0.05. BMSCs, bone marrow stem cells; SD, standard deviation.
Osteogenic Differentiation
The ALP activity levels of osteo-differentiated BMSCs, both in the presence or absence
of the devices, were higher than the undifferentiated ones. In particular, significantly
higher mean values were observed for BMSCs alone (+83%; p < 0.01) and for BMSCs incubated with the ThRevo-FT anchors (+39%; p < 0.05). A nonsignificant increase of ALP activity was observed in samples in contact
with JuggerKnot with respect to the control maintained in noninductive medium (p = 0.069). However, osteo-differentiated BMSCs cultured in the presence of the three
different devices showed lower levels of ALP activity in comparison with osteo-differentiated
BMSCs alone ([Fig. 2]).
Fig. 2 ALP activity levels of osteo-differentiated BMSCs after 14 days of culture, in the
presence or absence of the suture anchors. Values are normalized against the protein
content of each sample (data are expressed as the mean of two replicates ± SD). Control:
*p < 0.05; **p < 0.01; osteo-differentiated BMSCs: §
p < 0.05. ALP, alkaline phosphatase; BMSCs, bone marrow stem cells; SD, standard deviation.
As expected, the deposition of calcified matrix of differentiated BMSCs was higher
than one of the undifferentiated cells (+1,551%; p < 0.001). Similarly, BMSCs cultured with the devices produced higher levels of calcified
matrix in comparison with control samples, even if only tendency were found in the
statistical analysis, due to high variability in the samples. In particular, increases
of 1,238% (p = 0.057), 1,277% (p = 0.096), and +1,320% (p = 0.056) were observed for JuggerKnot, ThRevo-FT, and Cross-FT, respectively ([Fig. 3]).
Fig. 3 Quantification of calcified matrix deposition by Alizarin red S staining and extraction,
of osteo-differentiated BMSCs after 21 days of culture, in the presence or absence
of the devices (data are expressed as the mean of two replicates ± SD). Control: ***p < 0.001. BMSCs, bone marrow stem cells; SD, standard deviation.
Immune Response Activation
After 5 days of culture, cells were quantified by the Trypan blue staining method.
The PBLs counts in the samples incubated with the devices were similar to those from
the control group; hence, PBLs proliferation was not increased by the presence of
the devices. On the contrary, slight decreases in the number of PBLs were detected
in all the samples cultured in the presence of the devices ([Fig. 4]). Interleukin-2 content was determined by enzyme-linked immunosorbent assay after
5 days in culture and it resulted undetectable for all specimens.
Fig. 4 Peripheral blood leucocytes count from each culture condition (data are expressed
as the mean of two replicates ± standard deviation).
Discussion
Rotator cuff repair aims to restore the bone–tendon contact interface. Open and the
arthroscopic transosseous repair technique restore the bone–tendon interface without
the use of implant devices, other than the suture thread.[13] However, standard arthroscopic rotator cuff repair took advantage of implantable
devices, the suture anchors. These devices are within the bone–tendon interface and
they should not hinder tendon-to-bone healing process. Osteolysis and cysts, described
among the complications in the use of these devices, were supposed to have not only
mechanical causes but also local biological causes in the interaction of the anchors
with bone and soft tissues.[7]
[14]
Some previous studies focused on the different effects of these devices on the surrounding
cellular and tissue environment. Unfortunately, several variables are involved in
the modulation of healing processes and cellular signals, related to the implants
and to the patients as well. Zhang et al[15] reported that age and sex affect the ability of osteoblasts to produce bone. Furthermore,
injured tendons were found to have different collagen-type composition compared with
noninjured ones.[16]
Numerous modifications of the devices concerning material composition and coating
have been proposed to improve their osseointegration and tissue healing. A different
bone response, both in formation and in cells modulation, was found in vitro according
to chemical processing of the devices.[17]
Mazzocca et al[18] reported that a collagen-coated suture stimulates primary human osteoblasts and
tenocytes to proliferate, to adhere, and to differentiate, compared with a noncoated
suture. This was the first study that focused on the biocompatibility of suture anchors
made of different materials on the same cellular environment in vitro.
In this study, BMSCs cultured in the presence of all-suture, metal, and PEEK anchors
maintained their viability. Although cell viability resulted slightly decreased in
all the samples cultured in the presence of the devices, if compared with the control,
such reduction may have been provoked by the culture method. In fact, the direct contact
of the devices with the cells may have hampered the normal cell growth.
The presence of the devices in direct culture with BMSCs did not prevent their differentiation
ability toward the osteogenic lineage. Indeed, when maintained in osteogenic inductive
medium, all the samples were able to deposit higher amount of calcified matrix with
respect to the undifferentiated control. Similarly, ALP activity resulted higher in
all samples, compared with control cells in noninductive medium. Despite a comparison
with control cells in osteogenic medium showed a reduction in these parameters, the
entity of such a decrease is insubstantial because all the samples resulted positive
to calcified matrix deposition and ALP activity increased with respect to undifferentiated
controls.
None of the investigated devices was found to elicit an inflammatory response when
incubated with PBLs. The absence of PBL activation was confirmed by the lack of cell
proliferation and IL-2 production in all the analyzed samples, supporting the nonimmunogenicity
of the devices.
In conclusion, no relevant interference with normal cell functions was detected when
BMSCs or PBLs were cultured in direct contact with all-suture, metal, and PEEK suture
anchors. Further in vitro studies are needed to better evaluate the local effect of
suture anchors and the biological pathways to reduce complications and improve tendon
healing.
