著者は、骨軟骨プラグのラッピング過程で彼らの助けのためにUMCユトレヒトでの開発力学グループから氏のJeroenファンデンベルク氏Matthijs Wassinkに感謝の意を表したいと思います。この作品は、オランダの関節炎財団からの助成金によってサポートされていました。

注：ここで紹介するプロトコルは、最近の研究論文6、8、9の実験と計算手順から採用されています。プロトコルは、 図1に示されています。 死体の材料はユトレヒト大学の獣医学部の許可を得て収集しました。 1.サンプルおよびバス製造<ol><li>クールリン酸緩衝生理食塩水（PBS）を噴霧しながら過熱とその後の軟骨損傷を防止するために、カスタムメイドのドリルビット（ 図1）を用いて、死体ウマ大腿骨顆から円筒状の骨軟骨プラグ（8.5ミリメートルの直径）をドリル。 </li><li>オーバーレイ浴の横方向の拡散を最小限に抑えるためにプラスチック収縮スリーブと骨軟骨プラグを熱収縮。 <ol><li>目の軟骨と骨軟骨プラグをマウント電子のトップ当初、プラスチックシュリンクスリーブの内側とそれに熱風を吹き付けてください。熱関連の損傷を防ぐために、軟骨の表面に濡れた綿片を追加します。 </li></ol></li><li>別料金（ 例えば、650μL、420 mMの、イオキサグレート、分子量（Mw）= 1,269 Daの、電荷= -1）および中性（650μL、420ミリモル、イオジキサノール、MW = 1,550 Da）での溶質の有限浴を準備します。 </li><li>注射器を用いて軟骨の表面上に作製有限浴をロードし、RT（ 図2A）での実験中に蒸発を防ぐためにラップされたサンプル上にコルク栓を配置します。ニュートラル溶質輸送を研究するために、イオジキサノール槽を配置し、軟骨表面に負に帯電した溶質輸送場所のイオキサグレートバスを研究します。 </li></ol> 2.画像​​撮影・画像処理<ol><li>マイクロCTの電動ステージに取り付けられた特注ホルダにコルク栓を用いて単離し包まれたサンプルを置きます。置きトン彼は、造影剤溶液で覆われた軟骨表面が上を向いているように、試料。 </li><li>マイクロCT（40×40×40μmの3のボクセルサイズ、2分のスキャン時間、90キロボルトの管電圧及び180μAの管電流）軟骨、軟骨下プレートからなる視野を使用してスキャンし、finite-平衡状態（48時間）までのいくつかの時点で浴（ 図2A）に到達します。濃度値が時間とともに変化していない時に平衡状態が達成されます。 </li><li>メーカーのソフトウェアを使用して、関心領域（ROI）（ 例えば、分析）の位置決めを容易にするために、最初の画像に基づいて、異なる時点で3D画像を登録します。 </li><li>変換した3Dは、メーカーのソフトウェアを使用して、それらを処理する前に、2Dタグイメージファイル形式（TIFF）スタックにマイクロCT画像を再構成します。 </li><li>グローバルセグメント（ImageJの&gt;調整&gt;閾値）軟骨下骨と上から軟骨ソフトウェアのバスタブに横たわっ。 </li><li>前のステップで生成された軟骨のマスクを用いて（ImageJの&gt;&gt;測定分析）異なる時点での軟骨の平均グレー値を得ます。 </li><li>軟骨における初期浴濃度と初期の造影剤濃度を与えられ、溶質の実際の濃度を平均グレー値を変換するために、線形較正曲線を使用します。以前のデータは、グレー値が造影剤の濃度と線形関係を維持することをサポートします。 </li><li>実験の時点対溶質濃度をプロットします。 </li></ol> 3.計算モデル注：この問題に拡散実験境界条件に準拠し、（z軸に沿って）1Dに起こると仮定されます。したがって、ジオメトリが任意に作成することができます。 <ol><li>有限風呂に基づく軟骨マルチゾーンモデルを構築する：1）軟骨表面的なゾーンから成ります<html> （全軟骨厚さの20％）、中間ゾーン（全軟骨厚さの50％）と深いゾーン（全軟骨厚さの30％）12とFEBio 13、14（ 図2Bの2）有限浴）。 </li><li> FEBioでの軟骨および浴の異なるゾーンの機械的および物理的特性を割り当てます。ヤング率（10 MPa）を上層浴によって及ぼされる浸透圧に抵抗し、従って、過度の変形から軟骨を保護するのに十分高いと仮定しました。 <ol><li> 10 -3 mmでの透水率を使用して4 / Nsとシミュレーション8浴の0使用実際の溶質の拡散係数のポアソン比、9。 </li></ol></li><li>メッシュを生成する（8ノードトリリニアの六面体エレメント）との境界（ 図2B）の近くに絞り込みますクラス= &quot;外部参照&quot;&gt; 8、9。 </li><li>二相性-溶質モデル<ol><li>バスとそれに対応する有効圧力に初期溶質濃度を適用します。 9有効圧力、15の説明を見てください。 </li><li>異なる軟骨ゾーンにおいて所定の拡散係数に応じて時間曲線に対する溶質濃度を得るために過渡モードでモデルを実行します。 </li></ol></li><li>多相モデル 注：お風呂と組織との間の電気的な変動にはバスタブと組織の両方に2価の対イオンを加えることによって回避することができます。 <ol><li>定常状態モデルの場合：その所望の値にFCDを増加させながら、軟骨および上層浴中で同じ有効流体圧力および濃度を使用します。 </li><li>過渡モデルの場合：十分に高い浴中の溶質の拡散係数を維持することによって、よく攪拌有限風呂を作成します。注射します浴中に浴 - 空気界面からの溶質は、所望の濃度値に到達します。 </li><li>過渡：前のステップにおける所定の溶質濃度の境界条件を除去し、その実際の拡散係数に有限浴の拡散係数を元に戻します。 </li><li>異なる軟骨ゾーンに適用FirstClassディレクトリサービスと拡散係数に基づいて溶質濃度 - 時間曲線を得るために、モデルを実行します。 </li></ol></li><li> FEBio、MATLABインターフェイス<ol><li>自動的FEBioプロット濃度-時間曲線（FEBio-MATLABインタフェース）8、9でシミュレーションを実行するためにMATLABコードを開発。 </li><li> FEBio-MATLABインターフェイスを使用して、軟骨ゾーンの拡散係数とFirstClassディレクトリサービスを変更します。 FEBioでモデルを実行し、溶質濃度-時間曲線8,9を抽出します。 </li><li>実験で得られた溶質濃度 - 時間曲線の比較データおよび平均二乗誤差最小のルートに基づいて、異なる軟骨ゾーンで拡散係数とFirstClassディレクトリサービスのセットを得る（RMSE）8、9。 </li></ol></li></ol><img alt="図1" class="xfigimg" src="/files/ftp_upload/54984/54984fig1.jpg" /> 図1： 実験セットアップ。 A）カスタムメイドのドリルビットを使用してサンプル抽出手順。 B）マイクロCTイメージング手順は、拡散プロセスを監視します。 <a href="http://ecsource.jove.com/files/ftp_upload/54984/54984fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図2" class="xfigimg" src="/files/ftp_upload/54984/54984fig2.jpg" /> 図2： 回路図。A）実験計画。有限浴、表層、中間及び軟骨と関連メッシュの深い領域からなるB）マルチゾーン計算モデル。 <a href="http://ecsource.jove.com/files/ftp_upload/54984/54984fig2large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a>

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著者は、開示することは何もありません。

私たちは、関節軟骨全体で中性および充電溶質の拡散を研究するために、有限要素モデリング手法と組み合わせて実験プロトコルを提示しました。私たちの最近の研究によると、提案されたモデルは、正確に関節軟骨8、9の異なるゾーン間ニュートラル（二相性-溶質）の両方の輸送と負に帯電した（多相）溶質を記述することができます。広く、?...

<html>分子輸送は、関節の関節の恒常性に重要な役割を果たしている関節ための治療薬の配達軟骨と軟骨造影イメージング1、2、3。このような軟骨の統合と完全性、溶質の電荷およびサイズならびに軟骨と接触している浴の重量オスモル濃度および濃度のような因子は、トランスポートレート4、5、6に影響を及ぼし得ます。各ゾーンは、異なる濃度および主要細胞外マトリックス分子の配向から成るので、中性または荷電のいずれかの溶質の輸送は、関節軟骨のゾーン間で異なることができる、すなわち、プロテオグリカン（PG類）及びコラーゲンII型1、7、8、9、小娘= &quot;外部参照&quot;&gt; 10、11。より重要なことに、帯電した溶質の輸送は、関節軟骨8,9を横切って増加、細胞外マトリックス内の負の固定電荷を含むプロテオグリカンの濃度に大きく依存することができます。それらのパラメータ、特に固定電荷密度（FCD）、変形性関節症（OA）は、それによって軟骨を横切る拡散を研究することの重要性を意味する、進行するにつれて、軟骨を横断コラーゲン原線維と含水量変化の向きが変化を受けることができます。 今回の研究では、プロトコルは6、8、9を正確拡散の有限浴モデルに中性および充電溶質を使用して、さまざまな境界条件の下で拡散を調査するために提案され、以前に確立された実験と計算研究に基づきます。 T彼は、方法は、軟骨および高度な二相性-溶質および多相有限要素モデルでサポートされている有限浴を含むシステムのマイクロコンピュータ断層撮影（マイクロCT）で構成されている提案しました。これらのモデルは、関節軟骨の種々のゾーンを横切って中性と荷電分子ならびにFirstClassディレクトリサービスの拡散係数を得ることができます。これらのモデルを使用して、1は、軟骨および有限風呂を重ねる間の相互作用を調査するために使用することができ、拡散中性及び荷電した分子の挙動をよりよく理解を得ることができます。

<table><tbody><tr><td>Hexabrix</td><td>Guerbet</td><td>15HX005D</td><td>Negatively-charged contrast agent</td></tr><tr><td>Visipaque</td><td>GE healthcare</td><td>12570511</td><td>Nuetral contrast agent</td></tr><tr><td>PBS (Phosphate-buffered Saline)</td><td>Life technologies</td><td>10010023</td><td>Medium</td></tr><tr><td>micro-CT</td><td>Perkin Elmer</td><td></td><td>Monitoring diffusion</td></tr><tr><td>Freezing-point osmometer</td><td>Advanced instruments</td><td></td><td>Measuring solution osmolality</td></tr></tbody></table>

an experimental and finite element protocol to investigate the transport of neutral and charged solutes across articular cartilage

<html>変形性関節症（OA）は、関節軟骨および軟骨下骨の変性に関連している衰弱性疾患です。これは驚異的な化学的分解、 すなわちプロテオグリカン損失及びコラーゲン原線維破壊を経験するように関節軟骨の変性が実質的にその荷重支持機能を損ないます。 OA中に化学的損傷のメカニズムを調査するための一つの有望な方法は、外部の溶質に軟骨標本を公開し、分子の拡散を監視することです。健康な軟骨と比較して異なる拡散特性を作成する関節軟骨を横切って移動しながら、軟骨損傷（ すなわち、濃度及び本質的な巨大分子の構成）の程度は、外部溶質の衝突エネルギー損失と関連しています。本研究では、いくつかのステップで構成され、以前に開発された実験マイクロ-Cに基づいてされているプロトコルを導入します14px; &quot;&gt; omputed T omography（マイクロCT）及び有限要素モデリング第二相、溶質および拡散係数を得るために、多相有限要素モデルを適用することにより、続いてマイクロCTを使用して記録されて充電され、非荷電ヨウ素化分子の輸送。軟骨ゾーン間で電荷密度を修正しました。

ここで提供される代表的な結果は、以前の研究論文6、8、9、16から採用されています。 OAは、関節軟骨は有意な変化最も重要なGAGの損失、及びコラーゲン線維の損傷17、18、19</sup...

Watch this Scientific Journal Video about An Experimental and Finite Element Protocol to Investigate the Transport of Neutral and Charged Solutes across Articular Cartilage at JoVE.com

An Experimental and Finite Element Protocol to Investigate the Transport of Neutral and Charged Solutes across Articular Cartilage

我々は最近開発された実験と数値解析法を用いて関節軟骨全体に充電し、非荷電分子の輸送を調査するためのプロトコルを提案します。

実験および有限要素議定書は、関節軟骨全体で中性および荷電溶質の輸送を調査します

Osteoarthritis (OA) is a debilitating disease that is associated with degeneration of articular cartilage and subchondral bone. Degeneration of articular ...

an-experimental-finite-element-protocol-to-investigate-transport

Research

JoVE Journal

Bioengineering

6.2K ビュー.  Delft University of Technology (TU Delft). 我々は最近開発された実験と数値解析法を用いて関節軟骨全体に充電し、非荷電分子の輸送を調査するためのプロトコルを提案します。 

ビデオ: An Experimental and Finite Element Protocol to Investigate the Transport of Neutral and Charged Solutes across Articular Cartilage

Real-time Visualization and Analysis of Chondrocyte Injury Due to Mechanical Loading in Fully Intact Murine Cartilage Explants

Homeostasis of articular cartilage depends on the viability of resident cells (chondrocytes). Unfortunately, mechanical trauma can induce widespread chondrocyte death, potentially leading to irreversible breakdown of the joint and the onset of osteoarthritis. Additionally, maintenance of chondrocyte viability is important in osteochondral graft procedures for optimal surgical outcomes. We present a method to assess the spatial extent of cell injury/death on the articular surface of intact murine synovial joints after application of controlled mechanical loads or impacts. This method can be used in comparative studies to investigate the effects of different mechanical loading regimens, different environmental conditions or genetic manipulations, as well as different stages of cartilage degeneration on short- and/or long-term vulnerability of in situ articular chondrocytes. The goal of the protocol introduced in the manuscript is to assess the spatial extent of cell injury/death on the articular surface of murine synovial joints. Importantly, this method enables testing on fully intact cartilage without compromising native boundary conditions. Moreover, it allows for real-time visualization of vitally stained articular chondrocytes and single image-based analysis of cell injury induced by application of controlled static and impact loading regimens. Our representative results demonstrate that in healthy cartilage explants, the spatial extent of cell injury depends sensitively on load magnitude and impact intensity. Our method can be easily adapted to investigate the effects of different mechanical loading regimens, different environmental conditions or different genetic manipulations on the mechanical vulnerability of in situ articular chondrocytes.

We present a method to assess the spatial extent of cell injury/death on the articular surface of intact murine joints after application of controlled mechanical loads or impacts. This method can be used to investigate how osteoarthritis, genetic factors and/or different loading regimens affect the vulnerability of in situ chondrocytes.

リアルタイム可視化と完全にそのままマウス軟骨外植片の機械的荷重により軟骨損傷の解析

Homeostasis of articular cartilage depends on the viability of resident cells (chondrocytes). Unfortunately, mechanical trauma can induce widespread ...

生物工学

A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not regenerate very efficiently in vivo; and as a result, osteoarthritis leads to irreversible cartilage loss and is accompanied by chronic pain and immobility 1,2. Cartilage tissue engineering offers promising potential to regenerate and restore tissue function. This technology typically involves seeding chondrocytes into natural or synthetic scaffolds and culturing the resulting 3D construct in a balanced medium over a period of time with a goal of engineering a biochemically and biomechanically mature tissue that can be transplanted into a defect site in vivo 3-6. Achieving an optimal condition for chondrocyte growth and matrix deposition is essential for the success of cartilage tissue engineering. 
 In the native joint cavity, cartilage at the articular surface of the bone is bathed in synovial fluid. This clear and viscous fluid provides nutrients to the avascular articular cartilage and contains growth factors, cytokines and enzymes that are important for chondrocyte metabolism 7,8. Furthermore, synovial fluid facilitates low-friction movement between cartilaginous surfaces mainly through secreting two key components, hyaluronan and lubricin 9 10. In contrast, tissue engineered cartilage is most often cultured in artificial media. While these media are likely able to provide more defined conditions for studying chondrocyte metabolism, synovial fluid most accurately reflects the natural environment of which articular chondrocytes reside in.
 Indeed, synovial fluid has the advantage of being easy to obtain and store, and can often be regularly replenished by the body. Several groups have supplemented the culture medium with synovial fluid in growing human, bovine, rabbit and dog chondrocytes, but mostly used only low levels of synovial fluid (below 20&#37;) 11-25. While chicken, horse and human chondrocytes have been cultured in the medium with higher percentage of synovial fluid, these culture systems were two-dimensional 26-28. Here we present our method of culturing human articular chondrocytes in a 3D system with a high percentage of synovial fluid (up to 100&#37;) over a period of 21 days. In doing so, we overcame a major hurdle presented by the high viscosity of the synovial fluid. This system provides the possibility of studying human chondrocytes in synovial fluid in a 3D setting, which can be further combined with two other important factors (oxygen tension and mechanical loading) 29,30 that constitute the natural environment for cartilage to mimic the natural milieu for cartilage growth. Furthermore, This system may also be used for assaying synovial fluid activity on chondrocytes and provide a platform for developing cartilage regeneration technologies and therapeutic options for arthritis.

A 3D system of culturing human articular chondrocytes in high levels of synovial fluid is described. Synovial fluid reflects the most natural microenvironment for articular cartilage, and can be easily obtained and stored. This system thus can be used for studying cartilage regeneration and for screening therapeutics for treating arthritis.

滑液における培養ヒト関節軟骨細胞用の3Dシステム

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not ...

生物学

Characterization of Intra-Cartilage Transport Properties of Cationic Peptide Carriers

Several negatively charged tissues in the body, like cartilage, present a barrier to the targeted drug delivery due to their high density of negatively charged aggrecans and, therefore, require improved targeting methods to increase their therapeutic response. Because cartilage has a high negative fixed charge density, drugs can be modified with positively charged drug carriers to take advantage of electrostatic interactions, allowing for enhanced intra-cartilage drug transport. Studying the transport of drug carriers is, therefore, crucial towards predicting the efficacy of drugs in inducing a biological response. We show the design of three experiments which can quantify the equilibrium uptake, depth of penetration and non-equilibrium diffusion rate of cationic peptide carriers in cartilage explants. Equilibrium uptake experiments provide a measure of the solute concentration within the cartilage compared to its surrounding bath, which is useful for predicting the potential of a drug carrier in enhancing therapeutic concentration of drugs in cartilage. Depth of penetration studies using confocal microscopy allow for the visual representation of 1D solute diffusion from the superficial to deep zone of cartilage, which is important for assessing whether solutes reach their matrix and cellular target sites. Non-equilibrium diffusion rate studies using a custom-designed transport chamber enables the measurement of the strength of binding interactions with the tissue matrix by characterizing the diffusion rates of fluorescently labeled solutes across the tissue; this is beneficial for designing carriers of optimal binding strength with cartilage. Together, the results obtained from the three transport experiments provide a guideline for designing optimally charged drug carriers which take advantage of weak and reversible charge interactions for drug delivery applications. These experimental methods can also be applied to evaluate the transport of drugs and drug-drug carrier conjugates. Further, these methods can be adapted for the use in targeting other negatively charged tissues such as meniscus, cornea and the vitreous humor.

This protocol determines equilibrium uptake, depth of penetration and non-equilibrium diffusion rate for cationic peptide carriers in cartilage. Characterization of transport properties is critical for ensuring an effective biological response. These methods can be applied for designing an optimally charged drug carriers for targeting negatively charged tissues.

カチオンペプチドキャリアの軟骨内輸送特性の特性

Several negatively charged tissues in the body, like cartilage, present a barrier to the targeted drug delivery due to their high density of negatively ...

Labeling of Extracellular Vesicles for Monitoring Migration and Uptake in Cartilage Explants

Extracellular vesicles (EVs) are used in different studies to prove their potential as a cell-free treatment due to their cargo derived from their cellular source, such as platelet lysate (PL). When used as treatment, EVs are expected to enter the target cells and effect a response from these. In this research, PL-derived EVs have been studied as a cell-free treatment for osteoarthritis (OA). Thus, a method was set up to label EVs and test their uptake on cartilage explants. PL-derived EVs are labeled with the lipophilic dye PKH26, washed twice through a column, and then tested in an in vitro inflammation-driven&#160;OA model for 5 h after particle quantification by nanoparticle tracking analysis (NTA). Hourly, cartilage explants are fixed, paraffined, cut into 6 &#181;m sections to mount on slides, and observed under a confocal microscope. This allows verification of whether EVs enter the target cells (chondrocytes) during this period and analyze their direct effect.

Here, we present a protocol to label platelet lysate-derived extracellular vesicles to monitor their migration and uptake in cartilage explants used as a model for osteoarthritis.

軟骨外植木における移動と取り込みモニタリングのための細胞外小胞の標識

Extracellular vesicles (EVs) are used in different studies to prove their potential as a cell-free treatment due to their cargo derived from their ...

Mechanical Stimulation of Chondrocyte-agarose Hydrogels

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy this deficiency, several medical interventions have been developed. One such method is to resurface the damaged area with tissue-engineered cartilage; however, the engineered tissue typically lacks the biochemical properties and durability of native cartilage, questioning its long-term survivability. This limits the application of cartilage tissue engineering to the repair of small focal defects, relying on the surrounding tissue to protect the implanted material. To improve the properties of the developed tissue, mechanical stimulation is a popular method utilized to enhance the synthesis of cartilaginous extracellular matrix as well as the resultant mechanical properties of the engineered tissue. Mechanical stimulation applies forces to the tissue constructs analogous to those experienced in vivo. This is based on the premise that the mechanical environment, in part, regulates the development and maintenance of native tissue1,2. The most commonly applied form of mechanical stimulation in cartilage tissue engineering is dynamic compression at physiologic strains of approximately 5-20% at a frequency of 1 Hz1,3. Several studies have investigated the effects of dynamic compression and have shown it to have a positive effect on chondrocyte metabolism and biosynthesis, ultimately affecting the functional properties of the developed tissue4-8. In this paper, we illustrate the method to mechanically stimulate chondrocyte-agarose hydrogel constructs under dynamic compression and analyze changes in biosynthesis through biochemical and radioisotope assays. This method can also be readily modified to assess any potentially induced changes in cellular response as a result of mechanical stimuli.

The biosynthesis of cartilaginous extracellular matrix by chondrocytes can be affected by application of mechanical stimuli. This method describes the technique of applying dynamic compressive strains to chondrocytes encapsulated in 3D constructs and the evaluation of induced changes in chondrocyte metabolism.

軟骨アガロースゲルの機械的刺激

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy ...

A Friction Testing-Bioreactor Device for Study of Synovial Joint Biomechanics, Mechanobiology, and Physical Regulation

In primary osteoarthritis (OA), normal 'wear and tear' associated with aging inhibits the ability of cartilage to sustain its load-bearing and lubrication functions, fostering a deleterious physical environment. The frictional interactions of articular cartilage and synovium may influence joint homeostasis through tissue level wear and cellular mechanotransduction. To study these mechanical and mechanobiological processes, a device capable of replicating the motion of the joint is described. The friction testing device controls the delivery of reciprocal translating motion and normal load to two contacting biological counterfaces. This study adopts a synovium-on-cartilage configuration, and friction coefficient measurements are presented for tests performed in a phosphate-buffered saline (PBS) or synovial fluid (SF) bath. The testing was performed for a range of contact stresses, highlighting the lubricating properties of SF under high loads. This friction testing device can be used as a biomimetic bioreactor for studying the physical regulation of living joint tissues in response to applied physiologic loading associated with diarthrodial joint articulation.

The present protocol describes a friction testing device that applies simultaneous reciprocal sliding and normal load to two contacting biological counterfaces.

滑膜関節バイオメカニクス、メカノバイオロジー、物理調節の研究のための摩擦試験バイオリアクター装置

In primary osteoarthritis (OA), normal 'wear and tear' associated with aging inhibits the ability of cartilage to sustain its load-bearing and lubrication ...

Addressing Practical Issues in Atomic Force Microscopy-Based Micro-Indentation on Human Articular Cartilage Explants

Without a doubt, atomic force microscopy (AFM) is currently one of the most powerful and useful techniques to assess micro and even nano-cues in the biological field. However, as with any other microscopic approach, methodological challenges can arise. In particular, the characteristics of the sample, sample preparation, type of instrument, and indentation probe can lead to unwanted artifacts. In this protocol, we exemplify these emerging issues on healthy as well as osteoarthritic articular cartilage explants. To this end, we first show via a step-by-step approach how to generate, grade, and visually classify ex vivo articular cartilage discs according to different stages of degeneration by means of large 2D mosaic fluorescence imaging of the whole tissue explants. The major strength of the ex vivo model is that it comprises aged, native, human cartilage that allows the investigation of osteoarthritis-related changes from early onset to progression. In addition, common pitfalls in tissue preparation, as well as the actual AFM procedure together with the subsequent data analysis, are also presented. We show how basic but crucial steps such as sample preparation and processing, topographic sample characteristics caused by advanced degeneration, and sample-tip interaction can impact data acquisition. We also subject to scrutiny the most common problems in AFM and describe, where possible, how to overcome them. Knowledge of these limitations is of the utmost importance for correct data acquisition, interpretation, and, ultimately, the embedding of findings into a broad scientific context.

We present a step-by-step approach to identify and address the most common problems associated with atomic force microscopy micro-indentations. We exemplify the emerging problems on native human articular cartilage explants characterized by various degrees of osteoarthritis-driven degeneration.

原子間力顕微鏡を用いたヒト関節軟骨外植片のマイクロインデンテーションにおける実用的課題への対処

Without a doubt, atomic force microscopy (AFM) is currently one of the most powerful and useful techniques to assess micro and even nano-cues in the ...

Biotribological Testing and Analysis of Articular Cartilage Sliding against Metal for Implants

Osteochondral defects in middle-aged patients might be treated with focal metallic implants. First developed for defects in the knee joint, implants are now available for the shoulder, hip, ankle and the first metatarsalphalangeal joint. While providing pain reduction and clinical improvement, progressive degenerative changes of the opposing cartilage are observed in many patients. The mechanisms leading to this damage are not fully understood. This protocol describes a tribological experiment to simulate a metal-on-cartilage pairing and comprehensive analysis of the articular cartilage. Metal implant material is tested against bovine osteochondral cylinders as a model for human articular cartilage. By applying different loads and sliding speeds, physiological loading conditions can be imitated. To provide a comprehensive analysis of the effects on the articular cartilage, histology, metabolic activity and gene expression analysis are described in this protocol. The main advantage of tribological testing is that loading parameters can be adjusted freely to simulate in vivo conditions. Furthermore, different testing solutions might be used to investigate the influence of lubrication or pro-inflammatory agents. By using gene expression analysis for cartilage-specific genes and catabolic genes, early changes in the metabolism of articular chondrocytes in response to mechanical loading might be detected.

This protocol describes the preparation, biotribological testing, and analysis of osteochondral cylinders sliding against metal implant material. Outcome measures included in this protocol are metabolic activity, gene expression and histology.

インプラント用金属に対する関節軟骨滑走の生物化学検査と分析

Osteochondral defects in middle-aged patients might be treated with focal metallic implants. First developed for defects in the knee joint, implants are ...

医学

A Non-Invasive Method for Generating the Cyclic Loading-Induced Intra-Articular Cartilage Lesion Model of the Rat Knee

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely correlated with a history of articular cartilage damage and ligament avulsion. Surgical animal models of OA of the knee play an important role in understanding the onset and progression of post-traumatic OA and aid in the development of novel therapies for this disease. However, non-surgical models have been recently considered to avoid traumatic inflammation that could affect the evaluation of the intervention. 
In this study, an intra-articular cartilage lesion rat model induced by in vivo cyclic compressive loading was developed, which allowed researchers to (1) determine the optimal magnitude, speed, and duration of load that could cause focal cartilage damage; (2) assess post-traumatic spatiotemporal pathological changes in chondrocyte vitality; and (3) evaluate the histological expression of destructive or protective molecules that are involved in the adaptation and repair mechanisms against joint compressive loads. This report describes the experimental protocol for this novel cartilage lesion in a rat model.

Here, we present the cyclic loading-induced intra-articular cartilage lesion model of the rat knee, generated by 60 cyclic compressions over 20 N, resulting in damage to the femoral condylar cartilage in rats.

ラット膝の周期的負荷誘発関節内軟骨病変モデルを生成するための非侵襲的方法

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely ...

<meta charset="utf8"></head><body>ヒト関節軟骨からの軟骨原生細胞および軟骨細胞の単離

Isolation of Chondrocytes and Chondroprogenitors Using Fibronectin Adhesion and Migratory Assay

Chondroprogenitor cells (CPCs), recently identified as a distinct subpopulation, exhibit promise due to their mesenchymal properties, heightened chondrogenesis, and limited hypertrophic traits. The enrichment of progenitors is achieved through differential fibronectin adhesion and migration-based explant assays, with Fibronectin Adhesion Assay-derived Chondroprogenitors (FAA-CPs) and Migratory Chondroprogenitors (MCPs) demonstrating superior potential compared to chondrocytes. This article delves into the details of isolating resident cartilage-derived cells, namely chondrocytes and chondroprogenitors. While valuable insights from chondrocyte research contribute to our understanding of cartilage repair, ongoing efforts are directed toward the use of chondroprogenitors and exploring their potential as an alternative therapeutic approach. Additionally, this methodology article provides a detailed step-by-step protocol for isolating three specific cell types from cartilage: chondrocytes, FAA-CPs, and MCPs. By following standardized procedures, this protocol facilitates the successful extraction of these cell subtypes. Grounded in extensive research, the article focuses on the intricate techniques utilized in isolating the different subsets and the optimized culture conditions required to expand and maintain their cultures. The methodology encompasses enzymatic isolation of human articular cartilage-derived chondrocytes, differential fibronectin adhesion following sequential enzymatic digestion, and migration-based explant assays to obtain cartilage-resident cells.

<meta charset="utf8"></head><body>フィブロネクチン接着および回遊アッセイを用いた軟骨細胞および軟骨形成細胞の単離

This protocol details the isolation of chondrocytes, Fibronectin Adhesion Assay-derived Chondroprogenitors (FAA-CPs), and Migratory Chondroprogenitors (MCPs) from human articular cartilage. It covers enzymatic digestion, fibronectin adhesion, and migration-based assays for isolating and characterizing these cells.