Emilio Satoshi
Hara
a,
Masahiro
Okada
a,
Noriyuki
Nagaoka
b,
Takako
Hattori
c,
Letycia Mary
Iida
a,
Takuo
Kuboki
d,
Takayoshi
Nakano
e and
Takuya
Matsumoto
*a
aDepartment of Biomaterials, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama-shi, Okayama-ken, 700-8525, Okayama, Japan. E-mail: haraemilio@okayama-u.ac.jp; m_okada@cc.okayama-u.ac.jp; letycia.iida@gmail.com; tmatsu@md.okayama-u.ac.jp; Fax: +81-86-235-6669; Tel: +81-86-235-6666 Tel: +81-86-235-6667 Tel: +81-86-235-6665
bAdvanced Research Center for Oral & Craniofacial Sciences, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan. E-mail: nagaoka@okayama-u.ac.jp; Fax: +81-86-235-6669; Tel: +81-86-235-6734
cDepartment of Oral Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan. E-mail: hattorit@cc.okayama-u.ac.jp; Fax: +81-86-235-6669; Tel: +81-86-235-6646
dDepartment of Oral Rehabilitation and Regenerative Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan. E-mail: kuboki@md.okayama-u.ac.jp; Fax: +81-86-235-6680; Tel: +81-86-235-6682
eDivision of Materials and Manufacturing Science, Graduate School of Engineering, Osaka University, Osaka, Japan. E-mail: nakano@mat.eng.osaka-u.ac.jp; Fax: +81-6-6879-7512; Tel: +81-6-6879-7512
First published on 15th January 2018
Analysis of tissue development from multidisciplinary approaches can result in more integrative biological findings, and can eventually allow the development of more effective bioengineering methods. In this study, we analyzed the initial steps of mineral formation during secondary ossification of mouse femur based on biological and bioengineering approaches. We first found that some chondrocytes burst near the mineralized area. External factors that could trigger chondrocyte burst were then investigated. Chondrocyte burst was shown to be modulated by mechanical and osmotic pressure. A hypotonic solution, as well as mechanical stress, significantly induced chondrocyte burst. We further hypothesized that chondrocyte burst could be associated with space-making for mineral expansion. In fact, ex vivo culture of femur epiphysis in hypotonic conditions, or under mechanical pressure, enhanced mineral formation, compared to normal culture conditions. Additionally, the effect of mechanical pressure on bone formation in vivo was investigated by immobilization of mouse lower limbs to decrease the body pressure onto the joints. The results showed that limb immobilization suppressed bone formation. Together, these results suggest chondrocyte burst as a novel fate of chondrocytes, and that manipulation of chondrocyte burst with external mechano-chemical stimuli could be an additional approach for cartilage and bone tissue engineering.
Insight, innovation, integrationAnalysis of tissue development from multidisciplinary approaches can result in more integrative biological findings, and can eventually allow the development of more effective bioengineering methods. In this study, we first found that chondrocytes burst near the mineralized area in mouse femur epiphysis. Chondrocyte burst was shown to be modulated by osmotic and mechanical pressure. We further hypothesized that chondrocyte burst could be associated with space-making for mineral expansion. In fact, ex vivo culture of femur epiphysis in hypotonic conditions or under mechanical pressure enhanced mineral formation. Together, these results suggest chondrocyte burst as a novel fate of chondrocytes, and that manipulation of chondrocyte burst with external mechano-chemical stimuli could be an additional approach for cartilage and bone tissue engineering. |
Throughout the last few decades, numerous studies have investigated the cellular mechanisms of chondrogenesis, chondrocyte maturation and hypertrophy and mineral formation.2–5 Additionally, bioengineering techniques have been developed for manipulation of cells and tissues for cartilage synthesis and reconstruction.6,7 Of note, these studies have indicated that mineralization occurs inside the bioengineered cartilaginous pellets, mainly due to chondrocyte death (e.g., apoptosis) caused by hypoxic levels in the inner region of the cell aggregate (pellet). Therefore, bioengineered cartilaginous pellets have been used for analysis of chondrogenesis, as well as chondrocyte death and bone formation. Nevertheless, highly tunable manipulation of cartilage tissue-associated mineralization still remains a major difficulty. Understanding the physico-chemical and biological factors associated with or determining chondrocyte death and subsequent mineral formation is crucial for further improvement in bioengineering techniques for cartilage tissue synthesis and reconstruction, as well as for control of bone formation or even further bone marrow fabrication.
In this study, we first looked at the initial process of bone formation in the secondary ossification center of mouse femur epiphysis, in an attempt to understand the very initial events associated with chondrocyte fate and mineral formation. We first identified the initial minerals at post-natal day 5.5 (P5.5) by histological staining. Next, using femur epiphysis explants, we found that chondrocytes had burst near the mineralized area. We hypothesized that chondrocyte burst could be associated with mineral formation and expansion; thus, we used chemico-mechanical stimuli to induce chondrocyte burst, and to manipulate bone tissue formation inside the femur epiphysis in ex vivo experiments.
For analysis of the effect of osmotic pressure on mineral formation after induction of chondrocyte burst, isolated P6 femur epiphyses were incubated in PBS solutions with different osmotic pressure (hypertonic, isotonic (normal) and hypotonic) for 24 h, and then in the mineralization-inducing medium (DMEM/F12 supplemented with β-glycerophosphate) for an additional 2 days. PBS with 1.37 M NaCl, 2.7 mM KCl, 10 mM Na2HPO4·12H2O and 1.8 mM KH2PO4 was used as normal (isotonic) solution. PBS with no NaCl and 2.74 M NaCl were used as hypotonic and hypertonic solution, respectively. Since mineralization is highly sensitive to the amount of mineralization associated ions (i.e., calcium and phosphorus), DMEM with different concentrations was not used in the mineralization-inducing experiments in order to maintain the concentrations of mineralization-associated ions equal in the tissue. After incubation in the mineralization-inducing medium, samples were fixed with 4% PFA, and submitted to bone volume analysis by microCT. Micro-CT images of the collected epiphysis were obtained using a SkyScan 1174 compact micro-CT (SkyScan, Aartselaar, Belgium), at a resolution of 6.4 μm. Micro-CT sections were reconstructed to produce the final 3D images using Nrecon and CTVol SkyScan software. Bone volume in all epiphyses was analyzed under the same parameters using CTAn SkyScan software.
For analysis of bone formation upon mechanical pressure-induced chondrocyte burst, a rectangle plastic dish was used as a mold for agarose gel fabrication. The agarose sol was poured into the mold, and before complete gelation, the entire epiphyses were embedded in the gel. After complete gelation, plastic blocks 1 mm in thickness were inserted in between the plastic dish wall and the agarose gel, to induce pressure onto the epiphysis. Epiphyses were maintained under pressure for 3 h, and then removed from the gel and cultured in DMEM/F12 supplemented with β-glycerophosphate for an additional 48 h. Bone volume was further analyzed by micro-CT, under the same conditions described above.
Another observation from the time-lapse imaging was the extracellular space formed after chondrocyte burst (Fig. 2D), which was identical to the shape of the minerals observed in histological sections (Fig. 1B, arrowheads in the middle lower panel). Therefore, since hypertrophic chondrocytes were originally in close contact with each other, a decrease in chondrocyte volume due to chondrocyte death (burst or apoptosis14) could promote space for mineral expansion.
Next, in order to investigate the effect of osmotic pressure on chondrocyte burst-associated bone formation, the entire P6 epiphyses were cultured temporarily (24 h) in PBS with different osmotic pressure. As shown in Fig. 3B and C, there was a significantly higher amount of bone formation in the samples maintained in the hypotonic condition, compared to those in the normal or hypertonic condition.
Note that the aim for using FEA was not to calculate the accurate pressure distribution in the cartilage, but rather to analyze whether the pressure could concentrate in the region rich in hypertrophic cells (medial side). Therefore, a simple elastic 2D model was used in this study. It is also worth noting that we cannot justify even non-elastic 3D models in microscopic resolution due to the lack of parameters such as Young's modulus, diffusion coefficient and viscosity-related parameters of organic matrices, cell membranes and cytoplasms of normal and hypertrophic cells.
We then performed an in vivo experiment to reduce the body pressure onto the joints by immobilizing the lower limb with a plastic crutch fixed with a fast-acting cyanoacrylate adhesive at P3, before mineralization (Fig. 4A). Strikingly, we found no bone formation in the immobilized limb at P6, while bone development was normal in the contra-lateral control limb (Fig. 4B and C). Accordingly, at P7, bone volume was also decreased in the epiphysis of immobilized limbs, confirming a direct association between mechanical pressure and initial bone mineralization (Fig. 4D and E).
To analyze whether mechanical pressure could also induce chondrocyte burst, we used ex vivo slices of P6 epiphysis approximately 100 μm in thickness, and embedded the tissue in 2% agarose gel (Fig. 5A–C). The embedded cartilage samples were then submitted to different levels of mechanical pressure for 1 h, using an originally designed pressure device with a 3D-printed probe (Fig. 5A–C). For calculation of the pressure to be applied onto the epiphysis samples, the following values were considered. The body weight of a new born mouse is approximately 2 g (2 × 10−3 kg). Considering the gravitational field as 9.8 m s−2 and the joint area as 0.2 cm2, the pressure onto each joint would be (2 × 10−3 × 9.8 ÷ 0.2 ÷ 4 joints = 0.0245 kgf cm−2 = 2.4 kPa). The force applied onto the epiphysis-containing gel was 0.017 N (≈ 2.6 kPa) and 0.031 N (≈ 4.5 kPa) upon a compression of 250 μm and 500 μm, respectively. The authors used the distance in micrometers as a way to better control the pressure applied onto the epiphysis samples by using the micromanipulator. The agarose gel presented a linear elastic property under these experimental settings (Fig. 5D). As shown in Fig. 5E, the number of chondrocyte burst increased in a pressure-dependent manner, indicating a direct association of mechanical stress as a trigger for chondrocyte burst.
Finally, in an attempt to confirm whether mechanical pressure could be associated with mineral formation, the entire epiphyses were embedded in agarose and submitted to different mechanical pressures for 3 h in vitro (Fig. 6A). Afterwards, the epiphyses were removed from the agarose gel and cultured in β-glycerophosphate-supplemented DMEM/F12. As shown in Fig. 6B and C, mechanical pressure significantly induced mineral formation inside the epiphysis, in a pressure-dependent manner.
Taken together, these results indicate that mechanical stress is an important factor for chondrocyte burst and for in vivo mineral formation, suggesting that initial mineralization in the medial side of the femur epiphysis could be associated with mechanical stress-triggered chondrocyte burst.
A time-lapse imaging of ex vivo cartilage epiphysis samples showed that chondrocytes go through a bursting process. Here, we defined cell burst not as a complete destruction of the cells, but as a shrinkage of their volume. Previous studies have reported the chondrocyte bursting phenomenon, using monolayer cells20,21 or intact ex vivo cultured cartilage tissue.22 When chondrocytes were maintained in a hypotonic environment, there was a marked increase in chondrocyte volume without changes in cellular dry mass, and subsequently, the cells burst, with marked loss in dry mass.20,21 Accordingly, we herein demonstrated that a hypotonic condition markedly induced chondrocyte burst. Moreover, we showed that a hypotonic condition enhanced in vitro mineral formation inside the epiphyses.
To the authors’ knowledge, there is no study that has measured directly the osmotic pressure of tissues during bone development in vivo. Osmotic pressure in vivo can be supposed to be changed by several factors, such as by the invasion of negatively charged blood plasma proteins (e.g., albumin) in the extracellular region. Additionally, changes in the concentration of specific ions [e.g., calcium, phosphorus, hydrogen (pH)] could also be factors facilitating chondrocyte burst. It is known that cells make use of ATP-dependent ion pumps to control their osmolality in relation to the extracellular environment.15 Therefore, the activity of ATP or ion pump levels could be impaired or their expression levels decreased, in comparison to other cell types. In fact, loss of activity of ATP transporters, which affect intracellular ion concentration, is known to affect osmotic pressure and induce cell swelling.23 Subsequently, cell swelling could be an important phenomenon facilitating chondrocyte burst. Interestingly, a previous study using a single-cell photolysis experimental model showed that intracellular calcium rose prior to cellular membrane lysis induced by 632 nm laser light, and that this early rise in intracellular calcium was necessary for cellular membrane rupture.24 Treatment of cells with the channel blockers was effective in either decreasing or eliminating cell burst, even under lethal doses of photosensitizer and irradiation.24 Based on this report, we assumed that calcium could be one strong candidate factor facilitating chondrocyte burst. Accordingly, previous studies have demonstrated that fluid flow-induced shear stress or mechanical stress induced an increase in intracellular calcium possibly through mechanosensitive channels, and that these signals could be propagated to adjacent cells through gap-junctions.25–27 On the other hand, in vitro experiments have shown that high phosphate levels induce an increase in the expression of the hypertrophic chondrocyte marker, collagen type X, and also induce chondrocyte death, when associated with calcium.28 Thus, the dynamic changes in osmotic pressure, in particular after blood vessel invasion into the cartilage in femur epiphysis, after alkaline phosphatase activity inside chondrocytes (which would increase the intracellular phosphorus levels), and after initial calcium-phosphate formation in the extracellular matrix, could be altogether affecting intra- and extracellular ion concentrations and eventually could be directly or indirectly associated with chondrocyte burst and subsequent mineral formation.
Hypertrophic chondrocytes, which were first observed in the medial side of P4 and P5 epiphysis, present a 5-time increase in cell volume and also marked changes in their stiffness.16 A previous study reported that chondrocytes maintained in hypotonic conditions also present a significant increase in their volume, but a marked decrease in their viscoelastic properties associated with dissociation or redistribution of the F-actin cytoskeleton.21 In other words, osmotic pressure could be one potential factor associated with chondrocyte hypertrophy, and could facilitate chondrocyte burst by decreasing its viscoelastic properties. We then assumed that mechanical pressure could be another important factor inducing chondrocyte burst. In fact, mechanical pressure could significantly induce chondrocyte burst, and could also enhance mineral formation in the epiphysis. The pressure onto the joint (epiphysis) could induce a concentration of force in the medial side of the epiphysis, as simulated by FEA, and this pressure could then induce chondrocyte burst. Consequently, following the burst of chondrocytes, there could be permanent micro-deformation of the cartilage, which could in turn further enhance the concentration of mechanical pressure on this site. Of note, not only the anatomical morphology of the epiphysis (medial side being longer than the lateral side), but also biochemical modifications in hypertrophic chondrocytes, including the formation of intracellular vesicles1 (Fig. S3, ESI†), could be mechanically facilitating factors for cell burst in the medial side. Additionally, due to the highly charged and hydrated nature of the cartilage extracellular matrix, mechanical loading induces exudation of the interstitial water, resulting in dynamic changes in the interstitial osmolarity.20 Therefore, both mechanical and osmotic stress could be affecting simultaneously the chondrocytes and induce cell burst.
The present observations of chondrocyte burst may have an impact not only on developmental biology, but also on tissue engineering and bioengineering fields. External mechanical forces have been pointed out as important factors for in vitro synthesis of cartilage tissue, as these forces are known to induce the synthesis of ECM components by chondrocytes and strengthen the mechanical properties of the synthesized tissue.29 Nevertheless, application of intense mechanical stress to chondrocytes may potentially induce cell burst, and a deeper knowledge of the parameters and thresholds of the mechano-properties of chondrocytes, such as stiffness according to their differentiation stage (proliferative vs. hypertrophic chondrocytes), may direct novel methods for cartilage tissue engineering. On the other hand, mechanical stress has also been demonstrated to be an important factor regulating osteoblast and osteocyte activity, consequently affecting overall bone turnover. Previous studies have also shown that the stiffness of the surrounding microenvironment can control bone formation during primary endochondral ossification of mouse femur.2,3,8 Therefore, it may be possible that chondrocyte burst could be one intermediate event in these models of cartilage-to-bone formation, and manipulation of external mechanical properties of the surrounding environment could affect not only chondrocytes but also osteoblasts and osteocytes.
These results suggest that chondrocyte burst could be an event to promote space making for mineral expansion. More interestingly, chondrocyte burst could give origin to chondrocyte membrane nanofragments, which were shown to be nucleation sites for mineralization.30 In other words, chondrocyte death (e.g., apoptosis, burst) could not simply be a process for space-making, but could allow cellular components (e.g., phosphoproteins) to extravasate throughout the extracellular matrix, and could give origin to cell membrane nanofragments, which could become materials that promote mineral formation in the initial steps of osteoblast-independent mineralization in the femur epiphysis.30
In summary, we introduce the concept of chondrocyte burst as a novel fate of chondrocytes associated with initial mineral formation in femur epiphysis, and demonstrate that manipulation of chondrocyte burst with external mechano-chemical stimuli could be an additional approach for cartilage and bone tissue engineering.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c7ib00130d |
This journal is © The Royal Society of Chemistry 2018 |