Evaluation of five additives to mitigate toxicity of cryoprotective agents on porcine chondrocytes

Accepted Manuscript Evaluation of five additives to mitigate toxicity of cryoprotectant agents on porcine chondrocytes Kezhou Wu, Leila Laouar, Rachael Dong, Janet A.W. Elliott, Nadr M. Jomha PII:

S0011-2240(18)30302-X

DOI:

https://doi.org/10.1016/j.cryobiol.2019.02.004

Reference:

YCRYO 4059

To appear in:

Cryobiology

Received Date: 25 September 2018 Revised Date:

22 January 2019

Accepted Date: 25 February 2019

Please cite this article as: K. Wu, L. Laouar, R. Dong, J.A.W. Elliott, N.M. Jomha, Evaluation of five additives to mitigate toxicity of cryoprotectant agents on porcine chondrocytes, Cryobiology (2019), doi: https://doi.org/10.1016/j.cryobiol.2019.02.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Evaluation of Five Additives to Mitigate Toxicity of Cryoprotectant Agents on Porcine

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Chondrocytes

Affiliations: a Department of Surgery, University of Alberta, Edmonton, Alberta, T6G 2B7, Canada b Department of Chemical and Materials Engineering, University of Alberta, Edmonton, Alberta, T6G 1H9, Canada c Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, T6G 2R7, Canada d Department of Orthopaedic Surgery, First affiliated hospital of Shantou University Medical College, Shantou, Guangdong, 515300, China

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Authors: Kezhou Wua,d, Leila Laouara, Rachael Donga, Janet A. W. Elliottb,c, Nadr M. Jomhaa*

*Corresponding author. Address: 2D2.32 WMC Department of Surgery, University of Alberta Hospital, Edmonton, Alberta, Canada T6G 2B7, Fax: +17804072819. Email address: [email protected] (Nadr M. Jomha)

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Abstract: Cryoprotectant agents (CPAs) are used in cryopreservation protocols to achieve vitrification.

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However, the high CPA concentrations required to vitrify a tissue such as articular cartilage are a major

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drawback due to their cellular toxicity. Oxidation is one factor related to CPA toxicity to cells and tissues.

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Addition of antioxidants has proven to be beneficial to cell survival and cellular functions after

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cryopreservation. Investigation of additives for mitigating cellular CPA toxicity will aid in developing

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successful cryopreservation protocols. The current work shows that antioxidant additives can reduce the

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toxic effect of CPAs on porcine chondrocytes. Our findings showed that chondroitin sulfate, glucosamine,

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2,3,5,6-tetramethylpyrazine and ascorbic acid improved chondrocyte cell survival after exposure to high

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concentrations of CPAs according to a live-dead cell viability assay. In addition, similar results were seen

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when additives were added during CPA removal and articular cartilage sample incubation post CPA

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exposure. Furthermore, we found that incubation of articular cartilage in the presence of additives for 2

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days improved chondrocyte recovery compared with those incubated for 4 days. The current results

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indicated that the inclusion of antioxidant additives during exposure to high concentrations of CPAs is

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beneficial to chondrocyte survival and recovery in porcine articular cartilage and provided knowledge to

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improve vitrification protocols for tissue banking of articular cartilage.

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Keywords: Antioxidants; Additives; Cryoprotectant agents; Toxicity; Articular cartilage.

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Abbreviations:

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AA: ascorbic acid; CPA: cryoprotectant agent; CS: chondroitin sulphate; DMEM: Dulbecco's Modified Eagle Medium; Me2SO: dimethyl sulfoxide; EG: ethylene glycol; GlcN: glucosamine; PBS: phosphate buffered saline; PF-68: Pluronic F-68; PG: propylene glycol; TMP: 2,3,5,6-tetramethylpyrazine.

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Introduction

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Articular cartilage is a few-millimetres-thick tissue covering the bone end in articulating joints and it has

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very limited self-repair capacity [52]. Cartilage defects over 1 cm2 often progress into osteoarthritis if not

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properly treated [12, 32]. Cartilage allograft transplantation is an effective surgical procedure for large

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cartilage defects [25, 26]; however, it is limited by the availability of fresh donor joint tissue.

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Cryopreservation of articular cartilage would enable banking of these tissues for clinical transplantation

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[1, 10, 37, 38]. Vitrification, also called “ice-free” cryopreservation, is proven to be a useful method for

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long-term preservation of cells [10, 14, 38, 47, 50, 61] and our group developed a working vitrification

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protocol for osteochondral tissue of human knee articular cartilage with promising chondrocyte recovery

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and cellular metabolic function [38]. Vitrification can be achieved by using high concentrations of

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cryoprotectant agents (CPAs) and a rapid cooling rate [10, 19, 38, 47, 48, 50, 51]. However, one of the

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major obstacles is toxicity of high concentrations of CPAs to cells [3, 8, 18, 29, 36]. Dimethyl sulfoxide

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(Me2SO), ethylene glycol (EG) and propylene glycol (PG) are three CPAs commonly used in

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cryopreservation protocols [10, 14, 15, 38, 47, 48, 50, 51]. These CPAs are toxic to cells when used at

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high concentrations [3, 5, 18, 36]. There are several types of cell injury induced by CPAs, including

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membrane disruption and oxidative stress. Membrane disruption is induced by the high concentration of

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solutes outside the cell membrane, which is unavoidable in many current vitrification protocols [17, 43,

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45]. Oxidative stress, an oxidative reaction between cells and CPAs, is one important toxic event during

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CPA loading [42, 49, 53, 55]. Reducing oxidative reactions could improve cell recovery and benefit

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cryopreservation of cells and tissues [7, 29, 34, 55]. To mitigate oxidative injuries, some researchers that

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employ vitrification have found that inclusion of antioxidant additives as supplements in the CPA loading

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solution can benefit cell viability after exposure to high concentrations of CPAs [11, 13, 29, 34, 42].

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In the current study, five additive compounds including chondroitin sulphate (CS), glucosamine (GlcN),

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2,3,5,6-tetramethylpyrazine (TMP), ascorbic acid (AA) and Pluronic F-68 (PF-68) were examined.

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Among these, four additives: CS [28, 37], GlcN [4, 54, 57], TMP [20, 40, 44] and AA [6, 16] were

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proven to be antioxidants in previous studies. However, their protective effects on porcine articular

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cartilage in multi-CPA cocktail solutions remain unknown. CS is a sulfated glycosaminoglycan composed

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of a chain of alternating sugars and is an important structural component of articular cartilage [21]. CS

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was used previously in a vitrification study of human articular cartilage by our group and shown to be

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beneficial to cell viability in the rewarmed cartilage [1, 38]. GlcN is an amino sugar and is a precursor for

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glycosaminoglycan which is an important articular cartilage structural component [4, 54]. GlcN has been

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proven to beneficially influence cartilage structure and to alleviate arthritis pain in clinics [33, 54]. TMP

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is one of the alkaloids contained in Ligusticum wallichii and possesses anti-inflammatory and

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antioxidative activities [23, 31, 41] through inhibiting reactive oxygen species levels and increasing the

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antioxidant enzymes superoxide dismutase and catalase [23, 27, 56]. AA is an essential component in

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synthesis of collagen, a major component of cartilage extracellular matrix [6, 16]. AA is a highly effective

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antioxidant, acting to lessen oxidative stress and is an enzyme cofactor for the biosynthesis of many

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important biochemicals [2, 9, 16, 35, 46]. All these effects of CS, GlcN, TMP, or AA could decrease the

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severity of CPA oxidation effects on chondrocytes. Although PF-68 is not reported to be antioxidative; it

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is a non-ionic surfactant reported to be effective in maintaining cell membrane integrity which could

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reduce the disruption of chondrocyte cell membranes [24]. Oxidative stress on chondrocytes may lead to

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cell death, followed by degradation of the extracellular skeleton and structural collagen [30, 59].

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Elimination of oxidative stress has been proven to be beneficial to the cryopreservation of cells and

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tissues [11, 13, 28, 42, 53], because oxidation is one major reaction that can cause cell death. The

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objective of this study was to evaluate chondrocyte recovery after using these five additives during CPA

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exposure and removal. In this study, we hypothesized that the inclusion of additives could provide cell

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protection through the antioxidation effects on chondrocytes and by mitigating CPA toxicity due to the

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high concentration of CPAs. Investigating the effects of additives will aid in the development of

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successful vitrification protocols for articular cartilage.

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Materials and Methods

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Study design

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This study investigated the protective effects of several additives on articular cartilage chondrocytes after

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CPA exposure at specified temperatures determined by a CPA loading protocol. In this study, we

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evaluated the effects of five additive compounds in a stepwise multi-temperature, multi-CPA addition

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protocol [1, 38] using sexually mature porcine articular cartilage as a model. The objective of this study

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was to assess the abilities of these additives to mitigate chondrocyte cell toxicity during the exposure to

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high concentrations of CPAs at temperatures down to −10 °C, the final CPA loading temperature that

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would be used in a vitrification protocol before plunge cooling in liquid nitrogen.

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Sample source and preparation

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Hind legs with joints from sexually mature pigs aged over 54 weeks were obtained from 2 locations, a

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slaughter house in Wetaskiwin, Canada (Early Source), and from a deli store located in Edmonton,

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Canada (Late Source) (66 porcine hind legs resulting in 132 condyles were used in total). All joints were

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from meat-processing plants and no animals were specifically euthanized for this research. The joints

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from these two places were labelled as Early Source (< 6 hours post slaughter) and Late Source (> 24

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hours post slaughter) based on the sample collection times after slaughtering. Porcine joints from the

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Early Source were harvested and immersed in phosphate buffered saline (PBS) immediately then

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transported to the research laboratory within 6 hours on the same day, while joints from the Late Source

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were hung for 24 hours after slaughtering in the butchery and stored in a 4 °C fridge before transportation

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to the laboratory. All joints were immersed in PBS prior to dissection. An electric saw was used to split

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the condyles from the femora and all condyles with approximately 20 mm thick bone base were cleaned

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by incubation in sterile PBS plus antibiotics [100 units/ml penicillin, 100 µg/ml streptomycin and 0.25

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µg/ml amphotericin B (Gibco)] for 15 minutes under a biological safety cabinet.

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Condyles were

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randomly divided into the different experimental groups and incubated overnight in a complete medium

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plus antibiotics [DMEM complete: Dulbecco's Modified Eagle Medium F12 (Gibco) supplemented with

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10% heat-inactivated Fetal Bovine Serum, 100 units/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml

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amphotericin B and 1 mM sodium pyruvate] at 4 °C for less than 24 hours prior to CPA exposure.

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Cryoprotective agent cocktail solutions preparation

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Three fresh CPA cocktail solutions in DMEM were prepared the same day of the experiment into 175 ml

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final volume using the following concentrations of CPAs (M = molar): Solution One [3 M Me2SO + 3 M

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EG], Solution Two [3 M Me2SO + 3 M EG + 3 M PG], and Solution Three [3 M Me2SO + 3 M EG + 1 M

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PG]. Additives were added to the CPA cocktail solutions based on preset experimental conditions (see

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Experimental design). All the CPA cocktail solutions were stirred vigorously at 4 °C using an electronic

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stirrer for 30 minutes, then cooled to the desired temperature before starting CPA exposure (Solution One

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at 0 °C, Solution Two at −10 °C, and Solution Three at −10 °C).

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Stepwise CPA exposure protocol

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A stepwise CPA exposure protocol with multiple CPAs added at progressively lower temperatures was

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used to mimic CPA-toxicity-minimizing strategies according to our previous research [3, 18, 29, 36]. All

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condyles were paired and incubated stepwise in the three 175 ml CPA cocktail solutions: Solution One at

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0 °C for 90 min, followed by Solution Two, at −10 °C for 90 min, and last in Solution Three at −10 °C for

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130 min, then washed with 175 ml DMEM with or without additives at 4 °C for 45 min for CPA removal,

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then incubated with 100 ml DMEM with or without additives at 4 °C. Chondrocyte viability was assessed

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at four different time points: t0 = after overnight incubation before CPA exposure (fresh control), t1 = after

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the 3rd step CPA exposure followed by CPA removal in DMEM, t2 = after 2 days incubation at 4 °C post

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CPA removal, and t3 = after 4 days incubation at 4 °C post CPA removal (See Fig. 1). Percent cell

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viability in each experimental sample was normalized to the percent cell viability of its own fresh control

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at t0.

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Experimental design

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Experiment 1:

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supplemented with additives compared to a control group exposed to CPA solutions without

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additives.

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The objective of this experiment was to compare the effects of five additives, when added during the

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three-step CPA exposure, followed by CPA removal and a 4-day incubation without the presence of

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additives in the removal and incubation solutions. Porcine condyles were randomly divided into six

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groups (dissected joints from the two sources were pooled together, n = 14 condyles for each group). CPA

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cocktail solutions were prepared as described previously. Five additives were investigated separately in

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this study: 0.1 mg/ml CS (Sigma-Aldrich, St. Louis, Missouri), 400 µM GlcN (Medisca Pharmaceutique

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Inc., St-Laurent, Quebec), 400 µM TMP (Sigma-Aldrich, St. Louis, Missouri), 2000 µM AA (Sigma-

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Aldrich, St. Louis, Missouri), 0.1% PF-68 (vol/vol) (Gibco, Grand Island, New York), and compared with

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a control group exposed to CPA without additive. After the three-step CPA exposure for CPA permeation,

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condyles were washed in DMEM alone without additives for 45 min at 4 °C with shaking to remove the

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CPAs. A time of 45 min was expected to remove the majority of CPA based on our previous research

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[60]. Chondrocyte viability was measured at three time points: before CPA exposure (t0), after the 3rd step

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CPA exposure followed by CPA removal in DMEM (t1), and after 4 days incubation at 4 °C post CPA

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removal (t3).

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Chondrocyte viability in experimental groups exposed to CPA solutions

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Experiment 2: Chondrocyte viability determination by tissue freshness and the effects of AA

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and TMP when added during CPA exposure, removal, and DMEM incubation.

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The first objective of this experiment was to compare chondrocyte viability after CPA exposure in

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porcine condyles obtained from two sources. The second objective was to study the effect of TMP and

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AA separately during the CPA exposure period, post removal and after incubation for 2 and 4 days.

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Porcine joints collected from both the Early Source and the Late Source were dissected separately and

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condyles from the same source were randomly divided into four groups (n = 6 condyles for each group).

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In the AA +/− group, 2000 µM AA was added during CPA exposure but not during CPA removal or

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incubation. In the AA +/+ group, 2000 µM AA was added during CPA exposure, as well as during CPA

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removal and incubation. In the TMP +/− group, 400 µM TMP was added during CPA exposure but not

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during CPA removal or incubation. In the TMP +/+ group, 400 µM TMP was added during CPA

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exposure, as well as during CPA removal and incubation. Chondrocyte viability was measured at four

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time points: before CPA exposure (t0), after the 3rd step CPA exposure followed by CPA removal in

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DMEM (t1), after 2 days incubation (t2), and after 4 days incubation at 4 °C post CPA removal (t3).

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Table 1: Table of variables Study

1. CS

When Added

CPA exposure

Source

Pooled

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Additives

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Cell viability Assessment Timepoint 1. t1: Cell viability after CPA exposure followed by CPA removal

Goals of Study

Compare effects of additives during CPA exposure to control with no additive

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2. t3: Cell viability after 4-day DMEM incubation

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4. AA

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Experiment 2

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1. CPA exposure

1. Early Source

2. CPA removal

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2. TMP

1. t1: Cell viability after CPA exposure followed by CPA removal 2. t2: Cell viability

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1. Compare effects of sources 2. Compare effects of adding additive during CPA removal and

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3. DMEM incubation

after 2-day DMEM incubation 3. t3: Cell viability after 4-day DMEM incubation

3. Compare effects of 2-day incubation and 4-day incubation

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DMEM incubation

Chondrocyte viability assessment

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Eighty-five-µm-thick cartilage slices were cut from each condyle using a vibratome-1500 (The Vibratome

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Company, St. Louis, MO) filled with 500 ml pre-cooled PBS at 4 °C. Chondrocyte viability within slices

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was assessed by measuring cell membrane integrity with a dual stain using a fluorescent live cell nucleic

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acid stain (Syto 13; Molecular Probes) which is a cell permeant dye and propidium iodide (PI; Sigma)

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which is a membrane-impermeant dye that penetrates only into cells with disrupted cell membranes [6.25

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µM Syto 13 and 15.0 µM PI mixed in PBS]. After collecting slices, the remaining condyles were removed

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from the vibratome and washed with sterile PBS plus antibiotics for 15 minutes under sterile conditions,

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then stored in sterile DMEM complete (see sample source and preparation) at 4 °C until the next imaging

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time point. Slices were gently placed on glass slides and patted dry with Kimwipes. Each slice was

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overlaid with approximately 50 µl of stain mixture and covered with a coverslip, then incubated in the

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dark for 10 min. All slices were viewed under a Nikon inverted fluorescent microscope (model: ECLIPSE

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Ti-5) and imaged using a Nikon digital camera (model: DS-Fi2). The dual filters used to image all the

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slices in this study had the following spectra peak maxima: Excitation/Emission: 488 nm/503 nm and

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535 nm/617 nm. Chondrocyte viability within slices was determined by counting the numbers of green-

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stained (viable) cells and red-stained (non-viable) cells, using a custom-made software Viability 3.2 [39].

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Data analysis

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All data were analyzed using SPSS 20.0 software. A minimum 85% absolute cell viability in the fresh

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controls before CPA exposure (t0 cell viability) was used as the inclusion criterion to identify healthy

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tissue samples. Normalized cell viability of the experimental samples was determined according to the

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following formula:

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Normalized cell viability (%) =

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The numerical data are presented as the means ± SE. The p values are reported in the figure legends. In

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general, statistical significance in all the figures was indicated with asterisks respectively: * indicates p <

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0.05, ** indicates p < 0.01, *** indicates p < 0.001. The equality of variance for variables was

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determined by Levene’s test before multiple comparisons. The analysis of variance (ANOVA) with post

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hoc tests (Tukey test for multiple comparisons), paired t-tests and two-tailed independent t-tests were

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performed on sample cell viability under different experimental conditions depending on the sample

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configuration.



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Results

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Gross appearance of articular cartilage before and after CPA exposure and chondrocyte

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viability

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All condyles were harvested from porcine stifle joints. All condyles were removed from the porcine

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femur with an electric saw, then allocated into different experimental groups randomly. Healthy condyles

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had a smooth and white hyaline cartilage surface (Fig. 2A). Fluorescent microscopy on slices from fresh

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healthy articular cartilage showed most chondrocytes stained green indicating viable cells (Fig. 2B).

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However, when cartilage was exposed to CPA, a percentage of chondrocytes were stained red indicating

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cell death due to cell toxicity during CPA exposure (Fig. 2C). All samples with fresh control cell viability

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< 85% were excluded from this study to screen out unhealthy donors. The chondrocyte viability of the

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included condyles prior to CPA exposure ranged from 85 to 99 %.

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Chondrocyte viability assessment results

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Experiment 1: Comparison of chondrocyte viability in experimental groups exposed to CPAs

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with additives to that of a control group exposed to CPA without additives.

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Cartilage slices from condyles exposed to CPA solutions with additives showed significantly higher

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chondrocyte viability in TMP, AA, CS, and GlcN groups when compared to the control group exposed to

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CPA without additives (Fig. 3A). The TMP treated group showed the highest chondrocyte viability after

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CPA exposure followed by CPA removal (76.4 ± 9.1%) (Fig. 3A). After 4-day incubation in DMEM

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complete, chondrocyte viability in the CS and GlcN treated groups was significantly higher than the

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control group as shown in Fig. 3B. The CS treated group had the highest chondrocyte viability after

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incubation for 4-day post CPA removal (74.5 ± 10.7%) (Fig. 3B). However, the chondrocyte viability

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after 4-day incubation in TMP and AA groups was significantly lower when compared to the chondrocyte

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viability under the same conditions after CPA exposure followed by CPA removal (Fig. 3C).

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Experiment 2: Chondrocyte viability determination by tissue freshness and the effects of AA

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and TMP when added during CPA exposure, removal, and DMEM incubation.

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After exposure to CPA cocktail solutions supplemented with 2000 µM AA or 400 µM TMP, chondrocyte

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viability was significantly higher in the AA +/− group from the Early Source when compared to those

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obtained from the Late Source (Fig. 4A). After 2-day and 4-day incubation of condyles in DMEM

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complete without additives, both AA +/− groups and TMP +/− groups showed significantly higher

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chondrocyte viability in condyles from the Early Source when compared to those from the Late Source

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(Fig. 4A). The inclusion of AA in the DMEM wash solution during CPA removal significantly improved

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chondrocyte viability in AA +/+ group condyles from the Late Source compared to AA +/− group

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condyles from the Late Source washed without AA (Fig. 4B). When TMP or AA was added only during

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CPA exposure, condyles in the TMP +/− group from the Early Source, or condyles in the AA +/− group

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from the Late Source, showed significantly higher chondrocyte viability after 2-day incubation than that

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after 4-day incubation (Fig. 4C). When AA was added during the CPA removal and DMEM incubation,

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condyles in AA +/+ group from both the Early Source and the Late Source showed significantly higher

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chondrocyte viability after 2-day incubation than that after 4-day incubation (Fig. 4C). When incubating

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condyles for 4 days, condyles from the Late Source incubated with AA or TMP showed a significantly

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higher chondrocyte viability than those incubated without AA or TMP respectively (Fig. 4C).

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Discussion

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The main goal of this study was to investigate the effect of five additives on chondrocyte viability after

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exposure to high CPA concentrations. We hypothesized that CPA toxicity is linked to oxidative stress,

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which is induced during the exposure of cells to the CPAs. We firstly investigated the effects of CS, GlcN,

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TMP, AA and PF-68 on chondrocyte viability in full porcine condyles during exposure to high

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concentrations of CPAs. Condyles exposed to the stepwise CPA cocktail solutions supplemented with

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additives (CS, GlcN, TMP, or AA) showed higher chondrocyte viability compared to condyles in the

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control group exposed to CPA solutions without additives (Fig. 3B). Our findings showed decreased

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chondrocyte damage after CPA exposure in the presence of additives which confirmed the beneficial

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effect of these additives (CS, GlcN, TMP, or AA) during tissue exposure to high concentrations of CPAs.

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Cryopreservation has been proven to induce oxidative stress on the target cells both intracellularly and

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extracellularly during the freeze–thawing procedures [13, 42, 53]. Oxidative stress is one factor related to

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CPA toxicity that may aggravate cell sensitivity to high concentrations of CPA. The improvement in

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chondrocyte recovery of this study may be due to the antioxidative capacities of these compounds and this

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finding is consistent with previously reported work on the protective effects of antioxidants in

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cryopreservation [7, 9, 11, 22, 29, 38, 46]. However, unlike CS, GlcN, TMP, or AA, the use of PF-68

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group did not statistically benefit chondrocyte viability. PF-68 is a non-ionic surfactant known to protect

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chondrocytes from shear stress on cell membranes [24]. In this study, PF-68 did not have a beneficial

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effect, possibly due to low permeation of PF-68 into the articular cartilage matrix. Another variable

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investigated was 4-day incubation post CPA exposure. The high cell recovery after the 4-day incubation

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in medium post exposure to CPA plus additives in treated groups showed that chondrocytes remained at a

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higher survival rate compared to the control non-treated group (i.e. no additives) (Fig. 3C). Interestingly,

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the chondrocyte viability was significantly lower after 4-day incubation in DMEM complete without

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additives in groups treated with TMP or AA (Fig. 3C) when compared to their own chondrocyte viability

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immediately after CPA removal. The underlying mechanism of this cell loss may be due to the

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therapeutic effect timeframe of these two antioxidants (TMP and AA) as the additives were not present in

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the 4-day incubation solution. Further research on the compounds’ half-life elimination would help to

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understand their effects.

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We investigated sample freshness that may be a factor in cell recovery after CPA exposure. For instance,

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the stifle joints received from the Early Source were harvested from pigs soon after the animal was

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slaughtered and transported directly to our lab within 4–6 hours of death. Alternatively, joints received

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from the Late Source were harvested by meat plant employees and kept at 4 ⁰C for 24 hours in the fridge

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before the stifle joints were transported to our lab. Although the chondrocyte viability from both sources

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indicated a high cell membrane integrity (85–99%) before CPA exposure, the ability of chondrocytes to

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tolerate oxidative stress might be different. Subsequent investigation (Fig. 4A) documented that

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chondrocytes in fresher condyles obtained from the Early Source tolerated CPA exposure better than

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those obtained from the Late Source, with a significantly higher chondrocyte viability after CPA exposure.

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CPAs may be falsely assumed to be toxic to the cells if cell membranes were breached or damaged due to

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time after death factors, or if enzyme function was impaired for other reasons [8, 17]. We extended our

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work to the inclusion of additives during CPA removal and DMEM incubation using the two most

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promising additives—TMP and AA as per our current findings. Supplementation of media with additives

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during CPA removal and DMEM incubation improved chondrocyte recovery (Fig. 4B-C). A previous

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study using different concentrations of TMP demonstrated that TMP could inhibit interleukin-1β-induced

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iNOS expression and nitric oxide synthesis (NOS) in rabbit articular chondrocytes [31], while nitric oxide

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oxidation was one important reaction in the development of cartilage degradation during experimental

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osteoarthritis and chondrocyte apoptosis [30, 40]. Possible mechanisms of TMP and AA include

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antioxidant effects that might aid cell robustness during CPA exposure. Moreover, Yang et al. [58]

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reported a protective effect of TMP on oxygen and glucose deprivation induced brain microvascular

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endothelial cell injury via the Rho/Rho-kinase signaling pathway. Wang et al. [56] found that TMP could

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protect against glucocorticoid-induced apoptosis by promoting autophagy in mesenchymal stem cells and

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improve bone mass in glucocorticoid-induced osteoporosis in rats. Johnson et al. [35] reported AA could

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reduce DNA damage in H2O2-treated human adipose-derived mesenchymal stem cells, and Alam et al. [2]

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discovered that AA could prevent protein aggregation into oligomers and fibrils by inhibiting human

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insulin aggregation, and further protects against amyloid induced cytotoxicity. The higher chondrocyte

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viability in the AA and TMP groups indicates that these compounds are more likely to have a clinically

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significant effect and are good candidates for inclusion in cryopreservation protocols for cell preservation.

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In addition, we investigated the effects of 2-day incubation vs 4-day incubation. The chondrocyte viability

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after incubation for 2 days was higher than that after incubation for 4 days (Fig. 4C). This is partially

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consistent with a study by Hahn et al. [29] that reported that TMP had a significant beneficial effect on

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chondrocyte viability after exposure to 1.6 M glycerol also after a 2-day incubation period. That study did

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not test the 4-day incubation period. There is a concern that chondrocyte apoptosis may be the cause of

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the 4-day cell recovery decline and further investigation may confirm this. From the clinical perspective,

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it is possible that transplantation within the 2-day time frame after warming from vitrification would

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provide the cells the optimal environment for recovery.

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Conclusions

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CS, GlcN, TMP, or AA had a significant beneficial effect on chondrocyte viability after exposure to high

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concentrations of CPA. Fresh tissue contributed to higher cell recovery after CPA exposure and should be

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regarded as one factor in future experiments. CPA removal and incubation of condyles with the preferred

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additives (TMP or AA) improved chondrocyte recovery. Chondrocytes from articular cartilage incubated

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for a 2-day period had higher chondrocyte viability compared to chondrocytes from articular cartilage

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incubated for a 4-day period. Overall, using additives was beneficial for achieving higher chondrocyte

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viability after exposure to high CPA concentrations and this may be beneficial when developing

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cryopreservation protocols for articular cartilage in future research.

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Author contributions

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Experimental design: N.M. Jomha, J.A.W. Elliott, K. Wu, L. Laouar

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Performance of experiment: K. Wu, L. Laouar, R. Dong

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Evaluation of results: K. Wu, N.M. Jomha, J.A.W. Elliott

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Manuscript writing and review: K. Wu, L. Laouar, R. Dong, J.A.W. Elliott, N.M. Jomha

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Conflict of interests

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Dr. N.M. Jomha and Dr. J.A.W. Elliott are co-inventors on US and Canada patents for articular cartilage

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preservation: Jomha, N.M., McGann, L.E., Elliott, J.A.W., Law, G., Forbes, F., Torgabeh, A.A.,

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Maghdoori, B. and Weiss, A., University of Alberta, 2014. “Cryopreservation of articular cartilage”. US

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8,758,988; Canada 2,788,202.

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Acknowledgements

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This research was funded by the Edmonton Orthopaedic Research Committee (No. G522000020). Dr.

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J.A.W. Elliott holds a Canada Research Chair in Thermodynamics. K. Wu is funded by the Li Ka Shing

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Sino-Canadian Exchange Program between University of Alberta and Shantou University. We would like

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to offer special thanks to Dr. Locksley E. McGann for providing the Viability 3.2 software and valuable

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suggestions on the design of this study. We express our great appreciation to the Meat Shop at Pine

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Haven Colony and the Delton Sausage House & Deli in Edmonton for providing us with porcine joints.

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We acknowledge help provided by Ian McIntyre and Judy Unterschultz for sourcing the porcine joints

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from the Meat Shop at Pine Haven Colony. We thank Ethan Candler for his volunteer work in the lab

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during the summer.

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Figure 1: Description of multi-CPA stepwise exposure protocol: Chondrocyte viability of each condyle was determined by cell membrane integrity prior to any CPA exposure procedures (t0). Subsequently, the condyle was incubated in 175 ml of Solution One at 0 °C on ice and water for 90 minutes with shaking. Then the condyle was quickly taken out from Solution One and excess CPA surrounding the condyle was removed using paper towels. The condyle was then moved to 175 ml of Solution Two held in a stirred alcohol bath at −10 °C for 90 minutes, then removed quickly, dried and moved to 175 ml of Solution Three at −10 °C for 130 minutes. After the three steps of CPA loading, the condyle was washed in 175 ml DMEM with shaking for 45 min for CPA removal (t1). Condyles were then incubated in 100 ml sterile DMEM complete for 2 days (t2) to 4 days (t3) at 4 °C.

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Deep zone

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Figure 2: Gross appearance of the condyle morphology and chondrocyte viability based on Syto 13/PI membrane integrity staining: (A) In the fresh control condyles of porcine femora, the cartilage was smooth and intact; (B) In the fresh control condyle, fluorescent microscopy imaging of an 85-µm-thick slice showed mostly viable chondrocytes that stained green (Cross section, from left to right are the superficial to deep zones of articular cartilage, 100×); (C) In an experimental condyle exposed to CPAs, a percentage of cells died during CPA exposure, and dead cells were stained red (Cross section, from left to right are the superficial zone to deep zone of articular cartilage, 100×).

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A: After CPA Exposure Followed by CPA Removal

B: After 4-day Incubation Without Additives

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C: Comparison of CPA Removal With 4-day Incubation 90

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Figure 3: Chondrocyte viability in experimental groups exposed to CPAs with additives with results for the control group exposed to CPA without additives: Condyles from the two sources were mixed (n = 14). Condyles were exposed to the stepwise CPA protocol using five additives separately (CS: 0.1mg/ml; GlcN: 400µM; TMP: 400 µM; PF-68: 0.1% vol/vol; AA: 2000 µM) and a control group without additive. Condyles were then washed in DMEM alone for CPA removal and incubated in DMEM complete without additive. (A): After CPA exposure followed by CPA removal, chondrocyte viability was significantly higher in TMP, AA, CS, and GlcN groups compared to the control group (pCS-Control < 0.001, pGlcN-Control = 0.005, pTMP-Control < 0.001, pAA-Control < 0.001);

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(B): After 4-day incubation in DMEM complete, chondrocyte viability was significantly higher in CS and GlcN groups compared to the control group (pCS-Control = 0.001, pGlcN-Control = 0.007); (C): After 4-day incubation in DMEM complete, chondrocyte viability in TMP and AA groups were significantly lower compared to immediately after CPA exposure followed by CPA removal (pTMP = 0.025, pAA = 0.019).

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A: Comparison of Sources with AA and TMP ***

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C: Inclusion of Additives During 4-day Incubation

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Figure 4: Chondrocyte viability determination by tissue freshness and the effects of AA and TMP when added during CPA exposure, removal, and DMEM incubation: Four groups (AA +/−, AA +/+, TMP +/−, and TMP +/+, n = 6) were set with condyles from the Early Source (ES) and the Late Source (LS). In group AA +/－, 2000 µM AA was added during CPA exposure only; in group AA +/+, 2000 µM AA was added during CPA exposure, CPA removal, and DMEM incubation. In group TMP +/−, 400 µM TMP was added during CPA exposure only; in group TMP +/+, 400 µM TMP was added during CPA exposure, CPA removal, and DMEM incubation. (A): After CPA exposure followed by CPA removal, the AA +/− group showed a significantly higher chondrocyte viability in condyles obtained from the Early Source compared to those obtained from the Late Source (pAA-45min = 0.02). After 2 and 4day incubation, chondrocyte viability was significantly higher in the Early Source condyles treated with AA +/− or TMP +/− compared to the Late Source condyles (pAA-2day = 0.001, pAA-4day < 0.001, pTMP-2day = 0.008, pTMP-4day = 0.001);

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(B): When AA was added during CPA removal, chondrocyte viability was significantly higher in the AA groups of the Late Source condyles (pLS-AA = 0.006); (C): During the condyle incubation with or without AA or TMP, chondrocyte viability was significantly higher in condyles incubated for 2 days when compared to those incubated for 4 days (pES:AA+/+ = 0.012, pES:TMP+/− = 0.004, pLS:AA+/− = 0.007, pLS:AA+/+ = 0.038); After the 4-day incubation, chondrocyte viability was significantly higher in condyles from the Late Source incubated with AA or TMP when compared to those incubated without AA or TMP (pAA-4day = 0.049, pTMP-4day = 0.040).