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去細(xì)胞組織因保留天然細(xì)胞外基質(zhì)（ECM）的復(fù)雜成分和結(jié)構(gòu)，在組織再生方面有巨大潛力。我們通過(guò)調(diào)整人骨ECM來(lái)源的ECM水凝膠的骨粉大小和酶消化時(shí)間來(lái)控制其理化及生物學(xué)特性。減小骨粉尺寸、延長(zhǎng)消化時(shí)間可提高水凝膠的蛋白濃度、蛋白多樣性和凝膠強(qiáng)度。HBMSCs在骨粉水凝膠上培養(yǎng)后，成骨分化增強(qiáng)，證實(shí)這些水凝膠是骨再生的生物活性材料。

01研究?jī)?nèi)容簡(jiǎn)介

1、引言

組織工程利用細(xì)胞、生物活性分子和生物材料修復(fù)受損組織，具有廣闊的應(yīng)用前景。生物材料在組織工程中至關(guān)重要，它能夠提供微環(huán)境，并輸送能促進(jìn)再生組織中細(xì)胞功能的分子。諸多策略聚焦于開發(fā)模仿天然細(xì)胞外基質(zhì)（ECM）結(jié)構(gòu)和成分的生物材料。天然聚合物水凝膠，如膠原蛋白、海藻酸鹽、殼聚糖、透明質(zhì)酸和明膠，與 ECM 成分相似，且能形成纖維和多孔微觀結(jié)構(gòu)，調(diào)節(jié)細(xì)胞的黏附、增殖和分化等行為。但復(fù)制 ECM 中復(fù)雜的結(jié)構(gòu)和功能蛋白混合物，包括糖胺聚糖、膠原蛋白、蛋白聚糖和生長(zhǎng)因子，至今仍頗具挑戰(zhàn)。

組織脫細(xì)胞化成為制備和應(yīng)用生物材料的有效方法。去除天然組織中的細(xì)胞成分后，可保留 ECM 的復(fù)雜組成和結(jié)構(gòu)，還能降低免疫原性，減少炎癥反應(yīng)和疾病傳播。事實(shí)上，脫細(xì)胞 ECM 已被證實(shí)能促進(jìn)細(xì)胞行為和組織重塑。目前，動(dòng)物脫細(xì)胞基質(zhì)已用于多種臨床產(chǎn)品，如三維植入支架、二維薄片和粉末。最近，脫細(xì)胞 ECM 還被制成水凝膠。ECM 水凝膠主要由脫細(xì)胞 ECM 溶解或消化的蛋白質(zhì)組成，能在生理溫度和 pH 值下形成凝膠，可通過(guò)注射用于缺損部位，比 ECM 粉末分布更均勻。最常用的制備方法是用胃蛋白酶溶解脫細(xì)胞組織粉末，該酶能溶解   ECM 中的多種蛋白質(zhì)，多項(xiàng)研究表明，基于 ECM 蛋白質(zhì)的水凝膠保留了一定生物活性，能改善細(xì)胞增殖、分化和組織修復(fù)，自然也應(yīng)用于組織工程和再生醫(yī)學(xué)。

目前，關(guān)于牛和豬骨組織來(lái)源的脫細(xì)胞 ECM 水凝膠用于骨組織再生的研究有限，這些水凝膠已被證實(shí)具有增強(qiáng)細(xì)胞功能和促進(jìn)骨再生的能力。本研究探索利用人供體骨移植材料制備人 ECM 凝膠，以替代動(dòng)物組織來(lái)源的 ECM 水凝膠。無(wú)論組織來(lái)源如何，ECM 水凝膠都是去除細(xì)胞成分、免疫原性低的天然 ECM 材料。人體組織雖不易獲取，但免疫原性最低，異體移植是治療的金標(biāo)準(zhǔn)。動(dòng)物組織來(lái)源的 ECM 可能因某些動(dòng)物蛋白成分引發(fā)免疫反應(yīng)，如牛膠原蛋白、蛋白聚糖連接蛋白、聚糖抗原、豬內(nèi)源性逆轉(zhuǎn)錄病毒基因和 α - gal 表位。

在制備豬骨來(lái)源的 ECM 水凝膠時(shí)，脫礦脫細(xì)胞骨粉（<40   μm）在胃蛋白酶溶液中處理 1 小時(shí)。類似地，牛骨粉來(lái)源的 ECM 水凝膠需在胃蛋白酶溶液中消化 96 小時(shí)。然而，組織大小和消化時(shí)間對(duì) ECM 水凝膠的蛋白質(zhì)濃度、凝膠化、流變學(xué)和生物學(xué)特性的影響尚不明確。關(guān)鍵是，尚無(wú)針對(duì)人骨組織 ECM 水凝膠制備參數(shù)的研究。這對(duì)減少批次間差異（尤其是骨粉尺寸范圍廣的情況），以及通過(guò)優(yōu)化 ECM 蛋白質(zhì)比例最大化其再生功能至關(guān)重要。本研究專注于脫礦脫細(xì)胞人骨粉細(xì)胞外基質(zhì)來(lái)源的水凝膠，與傳統(tǒng)脫礦骨粉不同。

因此，本研究考察了顆粒大小和消化時(shí)間這兩個(gè)制備人骨 ECM 水凝膠的關(guān)鍵參數(shù)，對(duì)其理化和生物學(xué)特性的影響，以及在骨組織修復(fù)中的應(yīng)用潛力。人小梁骨組織取自骨科擇期手術(shù)的人股骨頭，并制備了不同尺寸（45 – 250 μm、250 –   1000 μm 和 1000 – 2000 μm）的脫礦人骨粉。這些骨粉經(jīng)胃蛋白酶處理 3、5 或 7 天。基于骨粉尺寸和消化時(shí)間，評(píng)估所得 ECM 水凝膠的凝膠化和流變學(xué)特性，用掃描電子顯微鏡和顯微計(jì)算機(jī)斷層掃描檢查水凝膠微觀結(jié)構(gòu)，通過(guò)測(cè)量細(xì)胞和蛋白質(zhì)濃度評(píng)估蛋白質(zhì)消化效率，用質(zhì)譜法評(píng)估水凝膠的蛋白質(zhì)組學(xué)特征，最后在人 ECM 水凝膠上檢測(cè)人骨髓基質(zhì)干細(xì)胞的黏附、鋪展、遷移、增殖和分化，以確定其生物學(xué)功能。

總之，本研究旨在探究骨粉尺寸和消化時(shí)間對(duì)人骨組織來(lái)源 ECM 水凝膠特性的關(guān)鍵影響，為開發(fā)有效的骨修復(fù)生物材料提供 ECM 理化特性方面的見解。

Fig. 1. Schematic illustration of ECM hydrogel derived from demineralized and decellularized human bone.Human trabecular bones were demineralized and decellularised. The digested extracellular matrix proteins were crosslinked, forming a gel to apply to a defect.

Fig. 2. Preparation process of ECM hydrogel derived from demineralized and decellularized human bone. (a) Human femoral heads from haematologically normal patients, both female and male, aged 65 ± 12 years old, were cut in half, and the trabecular bones collected and fragmented. (b) The fragments underwent demineralization in 0.5N HCl for 24 h, followed by lyophilization. The lyophilised powders were then sieved through stainless steel sieves to obtain powders in the ranges of 45–250 μm, 250–1000 μm, and 1000–2000 μm. (c) After decellularization in a Trypsin/EDTA solution, the powders were digested in a pepsin solution for 3, 5, and 7 days, respectively. The supernatant from the digestion solution was mixed with 0.1N NaOH, 10 × PBS, and 1 × PBS and incubated at 37 ?C for 1 h to induce gelation.

2.結(jié)果與討論

在生物材料和組織再生醫(yī)學(xué)領(lǐng)域，復(fù)制天然細(xì)胞外基質(zhì)（ECM）的特性一直備受關(guān)注。本研究旨在開發(fā)來(lái)源于人骨ECM的水凝膠，以應(yīng)用于骨科修復(fù)領(lǐng)域。我們假設(shè)ECM骨粉顆粒的大小和ECM消化的持續(xù)時(shí)間對(duì)ECM衍生蛋白質(zhì)的質(zhì)量和數(shù)量有重要影響，進(jìn)而影響所得水凝膠的流變學(xué)和物理化學(xué)性質(zhì)。通過(guò)考察人基質(zhì)細(xì)胞功能，我們探討了人骨ECM水凝膠在促進(jìn)骨組織修復(fù)方面的潛力。（圖2）

Fig. 3. Gelation and rheological characterizations of ECM hydrogels from various powder sizes and digestion time. (a) Turbidimetric gelation kinetics of ECM hydrogels. pH-neutralized ECM pepsin digests were added to the wells of a pre-warmed 96-well plate (37 ? C), and the absorbance at 405 nm was measured at 3-min intervals (n = 4). The values were normalized between 0 (the initial absorbance) and 1 (the maximum absorbance). (b) Photograph images of each gel after pH neutralization in inverted tubes before and after incubation at 37 ?C. The red line indicates the initial volume of the gel before inversion. (c) S (speed of gelation), tlag (time to start gelation), and t1/2 (time to reach 50 % maximum turbidity) were calculated based on turbidimetric curves. (d and e) Storage modulus (G′, open marks) and loss modulus (G″, closed marks) were monitored as hydrogels underwent an amplitude sweep of 0.1–100 % strain at a constant angular frequency. Data represent means ± standard deviation for n = 3. (f) Fourier transform infrared spectroscopy (FTIR) spectra of ECM digest compared with collagen with and without ECM buffer were analyzed. The spectrometer’s detection range was 399–4000 cm-1, and the data were measured with an interval of 0.96 cm–1 at room temperature. Statistical significance was determined using a two-way ANOVA test with Tukey’s multiple comparisons test (*p < 0.05 **p < 0.005 ***p < 0.0005****). The † symbol in-dicates a significant difference within the groups at the same digestion time). Data represent mean ± SD, N = 4.

3.1 骨粉大小和消化時(shí)間對(duì)水凝膠性質(zhì)的影響  

我們制備了多種類型的人骨ECM水凝膠，并研究了骨粉大小和消化時(shí)間對(duì)水凝膠凝膠化和流變性質(zhì)的影響。通過(guò)濁度法凝膠化動(dòng)力學(xué)分析，我們發(fā)現(xiàn)所有水凝膠的凝膠化動(dòng)力學(xué)均呈現(xiàn)S型模式。特別地，來(lái)自較小骨粉（45-250 μm）的水凝膠，無(wú)論消化時(shí)間如何，都表現(xiàn)出比來(lái)自較大骨粉（250-1000 μm和1000-2000 μm）的水凝膠更陡峭的斜率和更短的凝膠化時(shí)間（tlag和t1/2）。具體來(lái)說(shuō)，45-D3/D5/D7水凝膠在37℃孵育后約7分鐘內(nèi)即發(fā)生凝膠化，而250-D3/D5/D7和1000-D3/D5/D7水凝膠的凝膠化時(shí)間分別為16分鐘和20分鐘。 此外，我們還評(píng)估了水凝膠的流變特性。結(jié)果顯示，來(lái)自較小骨粉（45-250 μm）的水凝膠，特別是經(jīng)過(guò)5天和7天消化的水凝膠，其儲(chǔ)能模量（G'）顯著高于來(lái)自較大骨粉的水凝膠。這表明較小骨粉尺寸和較長(zhǎng)消化時(shí)間能夠溶解出更多種類的蛋白質(zhì)，從而生成具有更高機(jī)械強(qiáng)度的水凝膠。   為了測(cè)試水凝膠的穩(wěn)定性和體外降解情況，我們?cè)u(píng)估了水凝膠在PBS和膠原酶-PBS中的蛋白質(zhì)釋放量。結(jié)果顯示，在PBS中孵育后，約20-25%的蛋白質(zhì)從ECM水凝膠中釋放，且該比例在較長(zhǎng)時(shí)間內(nèi)保持穩(wěn)定。然而，在加入膠原酶-PBS溶液后，所有水凝膠中釋放的蛋白質(zhì)濃度迅速增加。值得注意的是，來(lái)自較小骨粉樣本的水凝膠表現(xiàn)出較慢的蛋白質(zhì)釋放曲線，表明其降解速度較慢。（圖3）

3.2 ECM水凝膠的凝膠化機(jī)制

我們研究了ECM水凝膠的凝膠化機(jī)制，并發(fā)現(xiàn)其與膠原蛋白提取物的凝膠化過(guò)程相似。通過(guò)中和ECM消化物的pH值和鹽濃度，并在37℃下孵育，可以誘導(dǎo)ECM水凝膠的形成。為了探究凝膠化過(guò)程中膠原蛋白的貢獻(xiàn)，我們使用傅里葉變換紅外光譜（FTIR）分析了中和ECM緩沖液對(duì)膠原蛋白和ECM消化前凝膠化學(xué)結(jié)構(gòu)的影響。結(jié)果顯示，ECM緩沖液中的NaCl和NaOH在膠原蛋白和ECM水凝膠的凝膠化過(guò)程中起著重要作用，它們通過(guò)形成膠原蛋白多肽鏈之間的連接來(lái)促進(jìn)凝膠化。（圖3）

Fig. 4. Microstructure analysis of human bone ECM hydrogels using scanning electron microscope (SEM) and micro-computed tomography (μ-CT). (a) SEM images of cross-sections of lyophilised ECM hydrogels were observed by SEM (scale bar = 300 μm), showing the presence of porous and fibrous structures in all types of ECM hydrogels. (b) 3-dimensional visualization of the ECM hydrogel structure using μ-CT demonstrates the porous nature of the hydrogels. (c) Measurement of pore sizes in ECM hydrogels using ImageJ software reveals no significant differences between the hydrogels (n = 3). Statistical significance was determined using a two-way ANOVA test with Tukey’s multiple comparisons test (*p < 0.05 **p < 0.005 ***p < 0.0005****). Data represent mean ± SD, N = 4.

3.3 水凝膠的微觀結(jié)構(gòu)

我們檢查了水凝膠的橫截面和三維結(jié)構(gòu)，發(fā)現(xiàn)所有水凝膠均表現(xiàn)出多孔和纖維狀結(jié)構(gòu)。纖維廣泛分布在孔隙中，這些孔隙相互連接，為細(xì)胞提供了良好的生長(zhǎng)環(huán)境。（圖4）

Fig. 5. Concentrations of DNA, total proteins, sGAG, collagen, BMP-2 and VEGF in ECM digests and histological staining images of ECM hydrogels. (a) Significantly high concentrations of proteins, including sGAG and BMP-2, were detected in the 45–250 μm powder and 7-day digested ECM digest, compared to other ECM digests (n = 4). Statistical significance was determined using a two-way ANOVA test with Tukey’s multiple comparisons test (*p < 0.05 **p < 0.005 ***p <0.0005****). The † symbol indicates a significant difference within the groups at the same digestion time). Data represent mean ± SD, N = 4. (b) ECM hydrogels were stained with haematoxylin and Alcian blue/Sirius red (scale bar = 100 μm). (c) Schematic illustration of the effects of bone powder size and digestion time on digesting ECM proteins.

3.4 ECM消化物中的細(xì)胞和蛋白質(zhì)含量  

我們量化了ECM消化物中DNA和蛋白質(zhì)的濃度，并發(fā)現(xiàn)總蛋白質(zhì)、sGAG和膠原蛋白的濃度隨著骨粉尺寸的減小和消化時(shí)間的增加而顯著增加。此外，我們還評(píng)估了ECM去細(xì)胞化的效果，并發(fā)現(xiàn)較小骨粉尺寸中的DNA濃度顯著高于較大骨粉，但隨著消化時(shí)間的增加，DNA濃度逐漸降低。這表明骨粉大小和消化時(shí)間對(duì)于從細(xì)胞外基質(zhì)中消除細(xì)胞至關(guān)重要。 為了可視化水凝膠中蛋白質(zhì)的分布，我們使用了特定染料進(jìn)行染色。結(jié)果顯示，水凝膠在膠原蛋白和GAG方面表現(xiàn)出顯著的染色，而細(xì)胞核染色可忽略不計(jì)。這表明水凝膠中含有豐富的膠原蛋白和GAG，這對(duì)于促進(jìn)細(xì)胞附著和生長(zhǎng)至關(guān)重要。（圖5）

Fig. 6. Comparison of the total matrisome subcategories of proteins in human bone ECM digests. (a and b) illustrate the percentages of proteins categorized under different matrisome subcategories in ECM digests. ECM digests derived from various sizes of bone powders after 5 days of digestion exhibit a significant impact on the protein proportions compared to those from shorter or longer incubation times. The dashed lines in (a) represent average percentages from the group. (c) The details and proportions of the proteins are listed in the heatmap.

Fig. 7. Total matrisome subcategories of proteins in human bone ECM digests from 45-D5, 250-D5, and 1000-D5. (a) presents the percentages of categorized proteins from 45-D5, 250-D5 and 1000-D5. (b) shows a Venn diagram depicting the percentage of matrisome subcategories of proteins in various sizes of bone powders after 5 days of digestion. Smaller bone powder exhibits a lower presence of collagens but a higher presence of other protein types. (c) The details of the proteins are listed in the table.

3.5蛋白質(zhì)組學(xué)分析

為了更深入地了解ECM水凝膠中蛋白質(zhì)的組成，我們進(jìn)行了蛋白質(zhì)組學(xué)分析。結(jié)果顯示，在ECM消化后，我們最多鑒定出了246種蛋白質(zhì)，包括核心基質(zhì)蛋白質(zhì)、基質(zhì)相關(guān)蛋白質(zhì)和非基質(zhì)蛋白質(zhì)。我們發(fā)現(xiàn)，骨粉大小和消化時(shí)間對(duì)ECM消化物中分類蛋白質(zhì)的比例有重要影響。具體來(lái)說(shuō)，較小骨粉尺寸和較長(zhǎng)消化時(shí)間能夠溶解出更多種類的蛋白質(zhì)，且這些蛋白質(zhì)在ECM水凝膠中的比例也更高。 此外，我們還發(fā)現(xiàn)，在較小骨粉尺寸和中等消化時(shí)間下，ECM水凝膠中含有更多種類的核心基質(zhì)蛋白質(zhì)、基質(zhì)相關(guān)蛋白質(zhì)和非基質(zhì)蛋白質(zhì)。這些蛋白質(zhì)在促進(jìn)細(xì)胞附著、生長(zhǎng)和分化方面可能發(fā)揮重要作用。 （圖5，6，7）

3.6 人骨ECM水凝膠對(duì)骨髓來(lái)源基質(zhì)細(xì)胞功能的影響

我們研究了人骨ECM水凝膠對(duì)骨髓來(lái)源基質(zhì)細(xì)胞（HBMSCs）功能的影響。通過(guò)一系列體外實(shí)驗(yàn)，我們發(fā)現(xiàn)人骨ECM水凝膠能夠顯著促進(jìn)HBMSCs的附著、鋪展、遷移、活力和增殖。這些結(jié)果表明，人骨ECM水凝膠為HBMSCs提供了一個(gè)良好的生長(zhǎng)環(huán)境，有助于其在體外擴(kuò)增和分化。 為了評(píng)估人骨ECM水凝膠在促進(jìn)骨組織再生方面的潛力，我們進(jìn)行了堿性磷酸酶（ALP）染色和ALP/DNA測(cè)定。結(jié)果顯示，與對(duì)照組

Fig. 8. Behaviour of HBMSCs on human bone ECM hydrogels. (a) HBMSCs were observed to attach and spread on the ECM hydrogels, irrespective of the hydrogel source from various bone powder sizes (scale bar = 2 μm). (b) ECM hydrogels were placed in the centre of tissue culture plate (TCP), and after seeding HBMSCs, their behaviours were observed using a time-lapse microscope. Some cells from the TCP migrated towards the ECM hydrogels (indicated by red arrows), while cells on the ECM hydrogels spread on top of the hydrogels (indicated by yellow arrows) (scale bar = 50 μm). (c and d) Cells stained with Calcein AM and DiD (green and red, respectively) were cultured in ECM hydrogels for 1, 3, and 7 days. (scale bar = 100 μm) (e) Cell viability and proliferation were evaluated using confocal imaging analysis and the WST-1 assay (n = 5). The cell population was noted to double within 7 days, and there was no significant difference in ECM hydrogels derived from various bone powder sizes. Statistical significance was determined using a one-way test with Tukey’s multiple comparisons test (*p < 0.05 **p < 0.005 ***p <0.0005****).(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

和膠原蛋白凝膠相比，ECM水凝膠上的HBMSCs表現(xiàn)出更高的ALP活性。這表明人骨ECM水凝膠能夠促進(jìn)HBMSCs向成骨細(xì)胞分化。為了進(jìn)一步驗(yàn)證這一結(jié)果，我們?cè)u(píng)估了HBMSCs在ECM水凝膠上培養(yǎng)2周和3周后早期成骨分化標(biāo)志基因（ALP、RUNX2和COL1A1）和晚期標(biāo)志基因（OCN）的表達(dá)水平。結(jié)果顯示，ECM水凝膠能夠上調(diào)這些基因的表達(dá)，特別是45-D7 ECM水凝膠。這表明45-D7 ECM水凝膠具有促進(jìn)HBMSCs早期成骨分化的潛力。 此外，我們還評(píng)估了HBMSCs在ECM水凝膠上的礦化能力。結(jié)果顯示，與膠原蛋白凝膠相比，ECM水凝膠能夠促進(jìn)更強(qiáng)的礦化反應(yīng)，特別是在細(xì)胞被封裝在凝膠內(nèi)部時(shí)。這表明ECM水凝膠的多孔和纖維狀結(jié)構(gòu)為細(xì)胞提供了一個(gè)有利于礦化的環(huán)境。   綜上所述，本研究表明人骨ECM水凝膠在促進(jìn)骨髓來(lái)源基質(zhì)細(xì)胞功能、早期成骨分化和礦化方面表現(xiàn)出優(yōu)異的性能。這些發(fā)現(xiàn)為人骨ECM水凝膠在骨科修復(fù)領(lǐng)域的應(yīng)用提供了有力支持。未來(lái)的研究將進(jìn)一步探索ECM水凝膠在體內(nèi)的骨修復(fù)效果，并確定其關(guān)鍵蛋白質(zhì)成分及其再生潛力。（圖8，9）

Fig. 9. Impact of human bone ECM hydrogels on HBMSCs osteogenic differentiation and mineralization. (a) Schematic showing the possible process of cell responses to ECM hydrogels. (b) HBMSCs morphology and alkaline phosphatase (ALP) staining density/colour differed notably between the 2D and 3D settings. (c) After 1 week of culture, ECM hydrogels significantly improved the ALP activities of HBMSCs, compared to tissue culture plate (ctrl) and collagen gels. (d) ECM hydrogels derived from bone powder sizes of 45–250 μm remarkably enhanced the early and late responses of HBMSCs differentiation compared to collagen gels and larger bone powder-sized ECM hydrogels. (e) Alizarin red S staining images of HBMSCs cultured for 3 weeks showed that ECM hydrogels promoted mineralization more effectively than collagen gel (scale bar: 200 μm). Notably, cells encapsulated within the ECM hydrogels exhibited a more robust mineralization response compared to cells seeded on top of the ECM gels. Statistical significance was determined using a one-way test with Tukey’s multiple comparisons test (*p < 0.05 **p < 0.005 ***p < 0.0005****). Data represent mean ± SD, N = 4.

3. 結(jié)論

源自人骨組織的脫細(xì)胞細(xì)胞外基質(zhì)（ECM）水凝膠在組織工程領(lǐng)域引起了廣泛關(guān)注，因其保留了天然的成分和結(jié)構(gòu)。本研究成功制備了來(lái)源于人體脫礦質(zhì)和脫細(xì)胞骨細(xì)胞外基質(zhì)的水凝膠。此外，我們重點(diǎn)闡述了骨粉粒徑和消化時(shí)間在人骨ECM水凝膠制備過(guò)程中的關(guān)鍵作用，以及它們對(duì)凝膠化和流變特性的調(diào)節(jié)，還有消化后蛋白質(zhì)的數(shù)量和組成豐富度的影響。

值得注意的是，較小的骨粉粒徑有助于消化更廣泛的ECM蛋白質(zhì)，這為骨組織工程應(yīng)用提供了具有顯著潛力的生物活性相關(guān)水凝膠。具體而言，骨粉粒徑的減小促進(jìn)了更多種類ECM蛋白質(zhì)的釋放和溶解，這些蛋白質(zhì)是構(gòu)成骨組織結(jié)構(gòu)和功能的重要成分。通過(guò)精確控制消化時(shí)間，我們可以進(jìn)一步優(yōu)化水凝膠的凝膠化過(guò)程，從而獲得具有理想流變特性的材料。這些水凝膠不僅保留了骨ECM的天然成分和結(jié)構(gòu)，還展現(xiàn)出了良好的生物相容性和生物活性，為骨組織修復(fù)和再生提供了新的可能。因此，本研究為人骨ECM水凝膠在骨組織工程領(lǐng)域的應(yīng)用奠定了堅(jiān)實(shí)的基礎(chǔ)。