原位可注射肝素-透明質(zhì)酸支架局部遞送轉(zhuǎn)化生長因子β1促進(jìn)軟骨形成

小夢想在努力 2020-02-26

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全層軟骨缺損在運動員中很常見，發(fā)生率高達(dá)36%。然而，由于軟骨細(xì)胞數(shù)量少和缺乏血供，軟骨組織自愈能力較差。因此，軟骨損傷的治療通常需要外科手術(shù)進(jìn)行自體軟骨細(xì)胞移植（ACI）。ACI技術(shù)是從關(guān)節(jié)的低負(fù)荷區(qū)域分離患者自身的軟骨細(xì)胞，然后在體外進(jìn)行擴(kuò)增，再將其植入軟骨缺損中。盡管該技術(shù)具有良好的臨床效果，但其價格昂貴。另外，成年軟骨細(xì)胞在擴(kuò)增過程中存在去分化現(xiàn)象，且不同患者間細(xì)胞成軟骨的能力存在不同，這些因素可能導(dǎo)致透明軟骨基質(zhì)形成的不同并產(chǎn)生不同的結(jié)果。為了克服這些局限性，人們開始尋找便于應(yīng)用的可注射材料，例如纖維蛋白膠等，進(jìn)行軟骨修復(fù)。然而，這些生物材料本身不具有誘導(dǎo)軟骨形成的能力。因此，研究人員探索了一系列包含生物活性物質(zhì)例如生長因子的誘導(dǎo)性生物材料，以指導(dǎo)軟骨形成。其中，轉(zhuǎn)化生長因子-β1（TGF-β1）已被廣泛用于軟骨組織工程中來誘導(dǎo)軟骨形成。然而有報道指出，高濃度的TGF-β1會引發(fā)副反應(yīng)，因此如何持續(xù)地、低劑量的緩釋從而達(dá)到更好的效果成為了現(xiàn)在研究的重點。

瑞士蘇黎世聯(lián)邦理工學(xué)院的Marcy Zenobi-Wong教授團(tuán)隊在轉(zhuǎn)谷氨酰胺酶因子XIII酶促交聯(lián)的透明質(zhì)酸水凝膠（HA-TG）中共價交聯(lián)肝素，然后實現(xiàn)對TGF-β1的緩釋。研究人員對比了HA-TG、HA-TG/Hep、HA-TG/Hep-TG三種水凝膠中TGF-β1的釋放行為。并且其中負(fù)載胚胎來源的人軟骨前體細(xì)胞（hCCs），研究這三種體系對hCCs軟骨形成的影響（圖1）。

Fig. 1. Schematic of the study design. (A) Acellular constructs were used for the characterization of the gelation behavior with rheology, as well as the release kinetics of heparin and of the initially-loaded TGF-β1 after gelation. (B) Human chondroprogenitor cells (hCCs) were encapsulated in either HA-TG, HA-TG with covalently bound heparin (HA-TG/Hep-TG) or HA-TG with non-covalently bound heparin (HA-TG/Hep). A comparison of the two different heparin incorporation methods was conveyed in terms of extracellular matrix production after 21?days. Further experiments comparing TGF-β1 administration method (loaded in the gel vs medium-supplemented) did not include HA-TG/Hep. Chondrogenesis was evaluated by characterizing matrix deposition via histology and biochemical assays, measuring chondrogenic genes expression with RT-qPCR as well as quantifying the stiffness with an unconfined compression test.

HA-TG比HA-TG/Hep-TG和HA-TG/Hep組表現(xiàn)出更快的釋放速度，而HA-TG/Hep與HA-TG/Hep-TG兩種體系差異不大。以上結(jié)果表明，HA-TG/Hep與HA-TG/Hep-TG兩種體系均可實現(xiàn)TGF-β1的緩釋。為了篩選中更適合軟骨形成的體系，研究人員研究了不同組中軟骨細(xì)胞外基質(zhì)（ECM）的形成情況。結(jié)果表明，未共價結(jié)合的肝素的加入并不能促進(jìn)軟骨的ECM沉積。因此，盡管共價結(jié)合和未共價結(jié)合的肝素均可實現(xiàn)TGF-β1的緩慢釋放，但共價結(jié)合肝素的HA-TG/Hep-TG在軟骨組織工程方面應(yīng)用前景更大。在下一步研究中，研究人員只選用了HA-TG和HA-TG/Hep-TG兩種體系（圖2）。

Fig. 2. Only covalent addition of heparin both efficiently retains TGF-β1 and promotes cartilaginous matrix deposition. (A) Gelation properties. Gelation curves obtained with a rheometer showed the storage modulus (unbroken lines) and the loss modulus (dotted lines) for two concentrations of HA-TG (2% in light blue and 1% in grey) and two heparin incorporation strategies (unbound in pink and crosslinked in dark blue, with 2% HA-TG for these conditions). N?=?3 for each condition. (B) Heparin release. Release curves of heparin (pink, n?=?3) and Hep-TG (dark blue, n?=?4) from the gels over 2?weeks measured with a DMMB assay. Student t test analysis for the last time point. (C) TGF-β1 release. Cumulative release curve of TGF-β1 from HA-TG/Hep gels (pink, n?=?3), HA-TG/Hep-TG gels (dark blue, n?=?3) and HA-TG gels (light blue, n?=?3) over 2?weeks. Student t test analysis for the last timepoint. (D) ECM deposition by encapsulated hCCs. GAGs and collagen type II production by hCCs encapsulated in either HA-TG with TGF-β1 supplemented in the medium (10?ng/ml), HA-TG/Hep or HA-TG/Hep-TG with TGF-β1 loaded in the gel at 800?ng/ml (20?ng/scaffold), n?=?4 for each condition. Scale bars:?200?μm (close-up) and 2?mm (inset). Histological controls are provided in Fig. B. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

研究人員對HA-TG和HA-TG/Hep-TG兩種體系中hCCs的細(xì)胞存活率及增殖進(jìn)行了研究。結(jié)果顯示，hCCs中多糖、I型膠原和II型膠原的表達(dá)在兩種體系中相似。體外培養(yǎng)第21天時，HA-TG/Hep-TG體系的壓縮模量比HA-TG低，但是仍在適用范圍內(nèi)（圖3）。

Fig. 3. hCCs chondrogenic potential is not affected by covalent addition of heparin to HA-TG, when cultured in TGF-β1-supplemented medium. (A) Schematic. (B) Cellular viability and proliferation assessed with (i) Live Dead staining, scale bar: 200?μm and (ii) Picogreen (fold change from d0 to d21) assay respectively. (C) GAG deposition. (i) Safranin O staining (n?=?3), (ii) DMMB quantification in papain-digested gels (n?=?4) and (iii) gene expression of aggrecan at d21 (n?=?3). (D) Collagen type I and II deposition. (i) Immunohistochemical stainings (n?=?3) and (ii) gene expression at d21 (n?=?3). (E) Mechanical characterization. Compression modulus after 21?days (kPa), n?=?4. For all histology images, scale bars: 200?μm (close-up) and 2?mm (inset).

進(jìn)一步地，研究人員研究了HA-TG/Hep-TG中TGF-β1劑量對負(fù)載的hCCs的存活率及增殖的影響?；钏廊旧Y(jié)果表明，HA-TG/Hep-TG中裝載的TGF-β1越多，hCCs存活率越高。并且，不同組中細(xì)胞形態(tài)也有所差別。細(xì)胞增殖結(jié)果也表明，在支架中hCCs未觀察到對TGF-β1明顯的劑量敏感性。當(dāng)支架中TGF-β1添加量≥5 ng時，其促進(jìn)hCC增殖的程度與在培養(yǎng)基中連續(xù)添加TGF-β1相似（圖4）。

Fig. 4. Initially loaded TGF-β1 supports hCCs viability and proliferation to a similar level as when continuously medium-supplemented, from a dose of 5?ng/scaffold. (A) Schematic. (B) Viability quantification. Live dead images quantified with Image J (n?=?3). “M” condition: 10?ng/ml TGF-β1 supplemented in medium. (C) Proliferation. Picogreen assay, n?=?4. ^****represents p?<?0.0001. (D) Viability assay. Live dead staining of scaffolds after 3?weeks of culture in serum-free medium. n?=?3. Scale bars: 100?μm.

研究人員也研究了HA-TG/Hep-TG中的TGF-β1劑量對軟骨ECM形成的影響。相對于培養(yǎng)基中添加TGF-β1，TGF-β1加到HA-TG/Hep-TG支架中會導(dǎo)致I型膠原和X型膠原基因表達(dá)降低。當(dāng)支架中TGF-β1添加量≥5 ng時，體外培養(yǎng)后可獲得具有明顯軟骨特征的組織，其具有典型的圓形細(xì)胞和均勻沉積的軟骨ECM。糖胺聚糖（GAG）的表達(dá)量隨TGF-β1的增加而增加。此外，I型膠原的形成也具有劑量依賴性。20ng組中II型膠原的表達(dá)量最高。當(dāng)支架中TGF-β1添加量≥5 ng時，支架中軟骨ECM的形成會使支架的壓縮模量增加?？傊Ъ苤?/span>TGF-β1對hCC的存活和軟骨形成具有劑量依賴性，最小閾值為5 ng，當(dāng)劑量為20 ng時達(dá)到穩(wěn)定。因此，研究人員選用了20 ng的劑量，來對比緩釋與突釋對軟骨形成的影響（圖5）。

Fig. 5. Initially loaded TGF-β1 supports homogeneous matrix deposition from a dose of 5?ng/scaffold with a positive dose response. (A) Collagen deposition. Immunohistochemical stainings of Collagen type II, I and X. Scale bars: 200?μm (close-up) and 2?mm (insert). N?=?3. (B-D) Collagen gene expression as fold increase compared to day 0 for (B) Collagen type II, (C) Collagen type I, (D) Collagen type X. “M” for medium supplementation. N?=?3 for each condition. (E) Stiffness. E moduli (also referred to as compressive moduli) obtained with an unconfined compression test, n?=?4. (F-H) GAG deposition. Safranin O staining for (F) loading conditions (dose / scaffold indicated on the top left of the insets) and (G) controls. Scale bars: 200?μm (close-up) and 2?mm (insert). N?=?3. (H) GAG quantification with DMMB assay on papain-digested gels, normalized to the background measured at day 0. N?=?4 for each condition.

因此，研究人員研究了TGF-β1的持續(xù)釋放對hCCs存活、增殖和軟骨ECM形成的影響。HA-TG與HA-TG/Hep-TG中細(xì)胞存活率相似，然而HA-TG/Hep-TG組中細(xì)胞增殖較快。此外，由于軟骨ECM形成的不同，HA-TG支架的硬度也顯著低于HA-TG/Hep-TG組。GAG的表達(dá)，在持續(xù)釋放TGF-β1支架中增加了2.6倍，而在突釋TGF-β1的組中增加了1.2倍，其增加量的不同部分是由于細(xì)胞增殖的差異導(dǎo)致的。II型膠原的表達(dá)趨勢與GAG類似。因此，研究人員認(rèn)為HA-TG/Hep-TG支架在加載GF用于軟骨損傷的可注射治療方面很有前途（圖6）。

Fig. 6. Sustained release of TGF-β1 is required for matrix deposition in vitro. (A) Schematics. (B) Viability, Proliferation and Mechanical properties. (i) Representative Live-dead staining images (scale bar: 200?μm) and (ii) plot of quantified values (n?=?3). (iii) Proliferation data obtained with a Picogreen assay, n?=?4. (iv) Elastic (E) modulus measured with an unconfined compression test, n?=?4. (C) GAG deposition. (i) Representative images of Safranin O stainings (n?=?3) and (ii) plotted values of quantified amounts of GAGs deposited, either normalized to the background at d0 (left) or to the amount of DNA (right), n?=?4. (iii) Plotted values of Aggrecan gene expression fold increase compared to d0. (D) Collagens deposition. Collagen type I and II representative immunohistological stainings (i) and plotted relative amount of gene expression relative to d0 (ii). For all histological images, scale bars: 200?μm (close-up) and 2?mm (insert).

本研究由瑞士蘇黎世聯(lián)邦理工學(xué)院的Marcy Zenobi-Wong教授團(tuán)隊完成，并于2019年9月發(fā)表于Acta Biomaterialia。

論文信息：

Clara Levinson, Mihyun Lee, Lee Ann Applegate, Marcy Zenobi-Wong. An injectable heparin-conjugated hyaluronan scaffold for local delivery of transforming growth factor β1 promotes successful chondrogenesis. Acta Biomaterialia 2019, 99: 168-180.