TWI649086B - Composition for repairing cartilage defects - Google Patents
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Abstract
本發明揭示一種用於修復軟骨缺陷的組合物。該組合物包含一骨髓濃縮物、一軟骨碎片或粉末、以及一滑液膜。同時亦揭示一種用於在個體中治療軟骨缺陷的方法。該方法包含施加本發明之組合物至該軟骨缺陷中，以及施加一纖維蛋白膠至該組合物上以覆蓋該軟骨缺陷。The invention discloses a composition for repairing cartilage defects. The composition comprises a bone marrow concentrate, a cartilage fragment or powder, and a synovial membrane. A method for treating cartilage defects in an individual is also disclosed. The method includes applying a composition of the present invention to the cartilage defect, and applying a fibrin glue to the composition to cover the cartilage defect.
Description
本發明關於一種用於修復軟骨缺陷的組合物。The invention relates to a composition for repairing cartilage defects.
運用自體軟骨細胞之組織工程已嶄露成為一種前景看好的軟骨再生方法。然而，有許多關於運用自體軟骨細胞的擔憂包含於採獲位置處造成傷害以及它們在單層擴張期間會有失去表現型的固有傾向。間質幹細胞 (Mesenchymal stem cells；MSCs)係經考慮的選擇細胞類型，因為它們具有可以一單一步驟程序分化成軟骨細胞的能力，而無須一生檢/培養期及分階再植入手術 (Fang HW, Biomed Eng 21:149–155 (2009))。由MSCs進行軟骨再生要成功的一個關鍵係正確調制細胞，使其分化至成軟骨路徑中並表現正常軟骨胞外基質 (ECM)。此外，ECM經認定會顯著參與引導MSCs分化程序 (Quesenberry PJet al. , 1 Stem Cells 16 (Suppl 1): 33–35 (1998); Philp Det al ., Stem Cells 23:288–296 (2005))。Tissue engineering using autologous chondrocytes has emerged as a promising method for cartilage regeneration. However, there are many concerns about the use of autologous chondrocytes, including the damage they cause at the harvest site and their inherent tendency to lose phenotype during monolayer expansion. Mesenchymal stem cells (MSCs) are considered to be selected cell types because they have the ability to differentiate into chondrocytes in a single step procedure without the need for a biopsy / culture period and staged reimplantation (Fang HW , Biomed Eng 21: 149–155 (2009)). A key to the success of cartilage regeneration by MSCs is to properly modulate cells to differentiate into the cartilage-forming pathway and to display normal cartilage extracellular matrix (ECM). In addition, ECM has been identified as significantly involved in guiding the differentiation process of MSCs (Quesenberry PJ et al. , 1 Stem Cells 16 (Suppl 1): 33–35 (1998); Philp D et al ., Stem Cells 23: 288–296 (2005 )).
最近，與自體軟骨細胞植入 (Autologous chondrocyte implantation；ACI) 相關的軟骨修復產品已經積極推銷至許多國家，以創造更多的生意。他們在優良組織操作 (Good Tissue Practice；GTP) 實驗室提供由一小軟骨生檢組織培養及擴張軟骨細胞的商業服務。然而，卻需要兩次手術且仍具有某些限制 (Buda Ret al. , Int Orthop. 2015 May;39(5):893-900; Pestka JMet al. , Am J Sports Med. 2014 Jan;42(1):208-15)。Recently, cartilage repair products related to Autologous chondrocyte implantation (ACI) have been actively marketed in many countries to create more business. They provide commercial services from a small cartilage biopsy tissue culture and expansion of chondrocytes in a Good Tissue Practice (GTP) laboratory. However, it requires two surgeries and still has certain limitations (Buda R et al. , Int Orthop. 2015 May; 39 (5): 893-900; Pestka JM et al. , Am J Sports Med. 2014 Jan; 42 (1): 208-15).
另一方面，若干已知已細胞為基礎的組合物需要一有效載體或封閉劑以固定至缺陷位置上。為求軟骨修復成功，移植物具備充足的初始機械穩定度係屬至高重要的 (Efe Tet al. , Knee Surg Sports Traumatol Arthrosc. 2012 Feb;20(2):210-5)。移植物必須在早期術後階段期間能承受得住關節活動期間活體內的力。必須有合適的固定以使移植物保持在缺陷區域並達到最佳的關節表面一致性。一般來說，在軟骨缺陷上會縫合一骨膜補片或膠原蛋白纖維網以避免細胞或載體/封閉劑的損失 (Slynarski Ket al. , Transplant Proc. 2006 Jan-Feb;38(1):318-9；Stone KRet al. , Arthroscopy. 2006 Mar;22(3):291-9；以及Christoph Erggeletet al. , Arch Orthop Trauma Surg, DOI 10.1007/s00402-009-0957-y (2009))。這也許會增加外科醫師的負擔並造成若干問題，如骨膜肥大 (Kreuzet al. , Osteoarthritis Cartilage. 2007 Dec;15(12):1339-47；Henderson Iet al ., Arthroscopy. 2006 Dec;22(12):1318-1324)。On the other hand, several known cell-based compositions require an effective carrier or blocking agent to fix to the defect site. For successful cartilage repair, it is of paramount importance that the graft has sufficient initial mechanical stability (Efe T et al. , Knee Surg Sports Traumatol Arthrosc. 2012 Feb; 20 (2): 210-5). The graft must be able to withstand the forces in the living body during joint movements during the early postoperative period. There must be proper fixation to keep the graft in the defect area and to achieve the best joint surface consistency. Generally, a periosteum patch or collagen fiber network is sutured over a cartilage defect to avoid the loss of cells or carriers / blockers (Slynarski K et al. , Transplant Proc. 2006 Jan-Feb; 38 (1): 318 -9; Stone KR et al. , Arthroscopy. 2006 Mar; 22 (3): 291-9; and Christoph Erggelet et al. , Arch Orthop Trauma Surg, DOI 10.1007 / s00402-009-0957-y (2009)). This may increase the burden on surgeons and cause problems such as periosteal hypertrophy (Kreuz et al. , Osteoarthritis Cartilage. 2007 Dec; 15 (12): 1339-47; Henderson I et al ., Arthroscopy. 2006 Dec; 22 ( 12): 1318-1324).
鑑於上述，仍然需要改善用於修復軟骨缺陷的組合物。In view of the foregoing, there remains a need for improved compositions for repairing cartilage defects.
在本發明中係非預期性地發現，一種包含骨髓濃縮物、軟骨碎片以及滑液膜的組合物在修復軟骨缺陷上展現極佳的特性。此外，非預期性地發現，施加在本發明之組合物上的纖維蛋白膠表現極佳的黏合性且不會如通常用於軟骨缺陷位置上隨時間而消散或損失，如此便不需要透過縫合骨膜或合成膜來覆蓋，填充有用於修復的以細胞為基礎的組合物的軟骨缺陷，此慣常的步驟。It was unexpectedly discovered in the present invention that a composition comprising bone marrow concentrate, cartilage fragments, and a synovial membrane exhibits excellent properties in repairing cartilage defects. In addition, it was unexpectedly found that the fibrin glue applied to the composition of the present invention exhibits excellent adhesion and does not dissipate or lose over time as is commonly used in cartilage defect sites, so that there is no need to go through sutures Periosteum or synthetic membranes are covered, filled with cartilage defects of cell-based composition for repair, this customary procedure.
因此，在一方面，本發明提供一種用於修復軟骨缺陷的組合物，其包含一骨髓濃縮物、一軟骨碎片或粉末、以及滑液膜。本發明之組合物可與一纖維蛋白膠組合使用。本發明之組合物使得新的一階段導向的軟骨修復手術成為可能，而無須細胞培養之GTP實驗室及製造新支架之GMP工廠。此可降低臨床可實行性的門檻並使從實驗室到臨床的表現獲益。Therefore, in one aspect, the present invention provides a composition for repairing a cartilage defect, which comprises a bone marrow concentrate, a cartilage fragment or powder, and a synovial membrane. The composition of the present invention can be used in combination with a fibrin glue. The composition of the present invention makes possible a new stage-oriented cartilage repair operation without the need for a GTP laboratory for cell culture and a GMP factory for manufacturing new scaffolds. This can lower the threshold of clinical feasibility and benefit from laboratory to clinical performance.
另一方面，本發明提供一種用於在個體中治療軟骨缺陷的方法，其包含施加本發明之組合物至該軟骨缺陷中，以及施加一纖維蛋白膠至該組合物上以覆蓋該軟骨缺陷。本發明之方法的一特徵在於，施加該組合物之後不需要骨膜或膜縫合之步驟來固定該組合物。In another aspect, the present invention provides a method for treating a cartilage defect in an individual, comprising applying a composition of the present invention to the cartilage defect, and applying a fibrin glue to the composition to cover the cartilage defect. A feature of the method of the present invention is that no periosteal or membrane suture step is required to fix the composition after applying the composition.
又一方面，本發明提供所述組合物，其用於在個體中治療軟骨缺陷的方法，該方法包含施加該組合物至該軟骨缺陷中，以及施加一纖維蛋白膠至該組合物上以覆蓋該軟骨缺陷。在本發明之一具體實施例中，該組合物係透過填入該軟骨缺陷來施加。根據本發明，該方法可不包含骨膜或膜縫合之步驟膜來覆蓋該軟骨缺陷。In another aspect, the present invention provides the composition for use in a method of treating a cartilage defect in an individual, the method comprising applying the composition to the cartilage defect, and applying a fibrin glue to the composition to cover The cartilage defect. In a specific embodiment of the invention, the composition is applied by filling the cartilage defect. According to the present invention, the method may not include a periosteum or a membrane of a membrane suture to cover the cartilage defect.
根據本發明，所述個體較佳為一哺乳類動物。該哺乳類動物包括但不限於人類、囓齒目動物、猴子、豬、狗及貓。更佳地，該個體為人類。According to the invention, the individual is preferably a mammal. The mammals include, but are not limited to, humans, rodents, monkeys, pigs, dogs, and cats. More preferably, the individual is a human.
在本發明中可使用來自具有與接受治療之個體相同或不同屬名之捐贈者的軟骨及滑液膜。Cartilage and synovial membranes from donors having the same or different genus names as the individual being treated can be used in the present invention.
應理解前述一般性說明及以下的詳細說明均僅為提供範例及解釋之用，而非用於限制本發明。It should be understood that the foregoing general description and the following detailed description are provided for the purpose of example and explanation only, and are not intended to limit the present invention.
在一方面，本發明提供一種用於修復軟骨缺陷的組合物，其包含一骨髓濃縮物、一軟骨碎片或粉末、以及一滑液膜。In one aspect, the present invention provides a composition for repairing a cartilage defect, comprising a bone marrow concentrate, a cartilage fragment or powder, and a synovial membrane.
本發明之組合物可與一纖維蛋白膠組合使用。纖維蛋白膠已經證實在許多矯形程序中十分有用；然而，其卻未在膝關節完全厚度軟骨缺陷中成功固定柔軟的材料，諸如細胞。The composition of the present invention can be used in combination with a fibrin glue. Fibrin glue has proven to be very useful in many orthopedic procedures; however, it has not been successful in fixing soft materials such as cells in full thickness cartilage defects of the knee joint.
另一方面，本發明提供所述組合物，其用於在個體中治療軟骨缺陷的方法，該方法包含施加該組合物至該軟骨缺陷中，以及施加一纖維蛋白膠至該組合物上以覆蓋該軟骨缺陷。在本發明之一具體實施例中，該組合物係透過填入該軟骨缺陷來施加。根據本發明，該方法可不包含骨膜或膜縫合之步驟來覆蓋該軟骨缺陷。In another aspect, the present invention provides the composition for use in a method of treating a cartilage defect in an individual, the method comprising applying the composition to the cartilage defect, and applying a fibrin glue to the composition to cover The cartilage defect. In a specific embodiment of the invention, the composition is applied by filling the cartilage defect. According to the present invention, the method may not include a periosteum or a membrane suture step to cover the cartilage defect.
此外，本發明提供一種用於在個體中治療軟骨缺陷的方法，其包含施加本發明之組合物至該軟骨缺陷中，以及施加一纖維蛋白膠至該組合物上以覆蓋該軟骨缺陷。In addition, the present invention provides a method for treating a cartilage defect in an individual, comprising applying the composition of the present invention to the cartilage defect, and applying a fibrin glue to the composition to cover the cartilage defect.
非預期地發現的是，施加在本發明之組合物上的纖維蛋白膠表現極佳的黏合性且不會如通常用於軟骨缺陷位置上隨時間而消散或損失，如此便不需要透過縫合骨膜或合成膜來覆蓋，填充有用於修復的以細胞為基礎的組合物的軟骨缺陷，此慣常的步驟。據此，在本發明之較佳具體實施例中，該方法不包含透過骨膜或膜縫合之步驟來覆蓋該軟骨缺陷。It was unexpectedly discovered that the fibrin glue applied to the composition of the present invention exhibits excellent adhesion and does not dissipate or lose over time as is commonly used in cartilage defect sites, thus eliminating the need to perforate the periosteum Or a synthetic membrane to cover, filled with cartilage defects of the cell-based composition for repair, this customary step. Accordingly, in a preferred embodiment of the present invention, the method does not include a step of covering the cartilage defect through a periosteum or a membrane suture.
根據本發明，所述個體較佳為一哺乳類動物。該哺乳類動物包括但不限於人類、囓齒目動物、猴子、豬、狗及貓。更佳地，該個體為人類According to the invention, the individual is preferably a mammal. The mammals include, but are not limited to, humans, rodents, monkeys, pigs, dogs, and cats. More preferably, the individual is human
本發明中所使用的｢骨髓濃縮物｣，其較佳的形式為(有核細胞的)細胞沉澱並可運用如離心之慣常方法由一骨髓濃縮物溶液來製備。該骨髓濃縮物溶液可透過以慣常方法移除血漿及紅血球碎片由骨髓抽吸物來製備，例如，該骨髓濃縮物溶液可由CellPoint™濃縮骨髓抽吸系統來製備 (美國，密蘇里州，ISTO科技)。其得到的骨髓濃縮物較骨髓抽吸物富含有核細胞。The "bone marrow concentrate" used in the present invention is preferably in the form of a (nucleated cell) cell pellet and can be prepared from a bone marrow concentrate solution by a conventional method such as centrifugation. The bone marrow concentrate solution can be prepared from bone marrow aspirate by removing plasma and red blood cell fragments by conventional methods. For example, the bone marrow concentrate solution can be prepared by CellPoint ™ concentrated bone marrow aspiration system (ISTO, Missouri, USA) . The bone marrow concentrate obtained is richer in nuclear cells than bone marrow aspirate.
在本文中所使用的術語｢有核細胞｣包括但不限於骨髓幹細胞、嗜中性球、淋巴球及單核白血球。The term "nucleated cells" as used herein includes, but is not limited to, bone marrow stem cells, neutrophils, lymphocytes, and monocytes.
根據本發明，該組合物可含有提供所欲之有核細胞數目的量的BMC。According to the invention, the composition may contain BMC in an amount that provides the desired number of nucleated cells.
例如，對於尺寸為深度1~3毫米及直徑約8.5毫米的軟骨缺陷 (即，一圓形缺陷)，所使用的骨髓濃縮物可包含104 -107 個有核細胞。亦即，該組合物可含有提供104 -107 個有核細胞的量的BMC。較佳地，該骨髓濃縮物包含105 -106 個有核細胞。在本發明之一具體實施例中，該骨髓濃縮物包含5 x 105 個有核細胞。For example, for a cartilage defect (i.e., a circular defect) having a size of 1 to 3 mm in depth and a diameter of about 8.5 mm, the bone marrow concentrate used may include 10 4 to 10 7 nucleated cells. That is, the composition may contain BMC in an amount that provides 10 4 to 10 7 nucleated cells. Preferably, the bone marrow concentrate comprises 10 5 to 10 6 nucleated cells. In one specific embodiment of the present invention, the bone marrow concentrate containing 5 x 10 5 nucleated cells.
在本發明之部分具體實施例中，該骨髓濃縮物的形式為細胞沉澱且實質上由源自骨髓抽吸物的有核細胞所構成。例如，有核細胞占本發明中所使用之骨髓濃縮物重量的至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%、或至少99%、或更多。In some embodiments of the present invention, the bone marrow concentrate is in the form of a cell pellet and is substantially composed of nucleated cells derived from bone marrow aspirate. For example, nucleated cells make up at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 90% by weight of the bone marrow concentrate used in the present invention. 98%, or at least 99%, or more.
軟骨碎片或粉末可由來自包括但不限於膝關節、髖關節及踝關節之關節的軟骨組織來製備。在部分具體實施例中，軟骨粉末係無細胞軟骨粉末，例如，Cartiform®。根據本發明，軟骨碎片的尺寸範圍較佳為從1毫米x1毫米至3毫米x3毫米。在一具體實施例中，軟骨碎片的尺寸為1-2毫米x 2-3毫米。Cartilage fragments or powder can be prepared from cartilage tissue from joints including, but not limited to, knee, hip, and ankle joints. In some embodiments, the cartilage powder is acellular cartilage powder, such as Cartiform®. According to the invention, the size of the cartilage fragments is preferably from 1 mm x 1 mm to 3 mm x 3 mm. In a specific embodiment, the size of the cartilage fragments is 1-2 mm x 2-3 mm.
對於尺寸為深度1~3毫米且直徑約8.5毫米的軟骨缺陷 (即，一圓形缺陷)，可使用30-65毫克之軟骨碎片或粉末。例如，所述組合物可包含60毫克之軟骨粉末。For cartilage defects (i.e., a round defect) having a depth of 1 to 3 mm and a diameter of about 8.5 mm, cartilage fragments or powders of 30-65 mg can be used. For example, the composition may include 60 mg of cartilage powder.
本發明之組合物中所使用的滑液膜可為碎片形式。較佳地，所述滑液膜係尺寸範圍從1毫米x1毫米至3毫米x3毫米的滑液膜碎片。在本發明之一具體實施例中，碎片的尺寸為約2毫米x 3毫米。The synovial film used in the composition of the present invention may be in the form of chips. Preferably, the size of the synovial membrane is from 1 mm x 1 mm to 3 mm x 3 mm. In a specific embodiment of the invention, the size of the fragments is about 2 mm x 3 mm.
對於尺寸為深度1~3毫米且直徑約8.5毫米的軟骨缺陷 (即，一圓形缺陷)，可使用30-65毫克之滑液膜。例如，所述組合物可包含60毫克之滑液膜。For cartilage defects (i.e., a round defect) having a size of 1 to 3 mm in depth and a diameter of about 8.5 mm, a synovial membrane of 30-65 mg can be used. For example, the composition may include 60 mg of a synovial film.
本發明中可使用自體骨髓濃縮物或來自具有與待接受治療之個體相同或不同屬名之捐贈者的非自體骨髓濃縮物。Autologous bone marrow concentrates or non-autologous bone marrow concentrates from donors with the same or different genus names as the individual to be treated can be used in the invention.
本發明中可使用來自具有與待接受治療之個體相同或不同屬名之捐贈者的軟骨及滑液膜。Cartilage and synovial membranes from donors with the same or different genus names as the individual to be treated can be used in the present invention.
根據本發明，組合物可透過均勻混合一骨髓濃縮物、一軟骨碎片或粉末、及一滑液膜來製備。According to the present invention, the composition can be prepared by uniformly mixing a bone marrow concentrate, a cartilage fragment or powder, and a synovial membrane.
例如，包含104 -107 個有核細胞之骨髓濃縮物可與40-100毫克軟骨碎片或粉末及40-100毫克滑液膜混合以形成用於軟骨缺陷修復之組合物。該組合物可進一步與0.2-1毫升之生物相容載體混合。具有普通技能者可根據欲進行修復之軟骨缺陷尺寸輕易地調整實際的量。在本發明之一具體實施例中，該組合物係透過混合包含約5 x 105 個有核細胞之骨髓濃縮物與60毫克軟骨碎片及60毫克滑液膜碎片、進一步與0.2毫升之纖維蛋白膠混合來製備。For example, a bone marrow concentrate containing 10 4 to 10 7 nucleated cells can be mixed with 40-100 mg of cartilage fragments or powder and 40-100 mg of synovial membrane to form a composition for cartilage defect repair. The composition may be further mixed with 0.2-1 ml of a biocompatible carrier. Those with ordinary skills can easily adjust the actual amount according to the size of the cartilage defect to be repaired. In one specific embodiment of the present invention, the composition comprises about line 5 x 10 5 th bone marrow mononuclear cells of a concentrate with 60 mg and 60 mg fragments of cartilage synovial membrane fragments through mixing, with a further 0.2 ml of fibrin Glue to prepare.
根據本發明，所述組合物可包含0.1-1重量百分比之BMC、30-60重量百分比之軟骨碎片或粉末、及30-60重量百分比之滑液膜。According to the present invention, the composition may include 0.1-1 weight percent of BMC, 30-60 weight percent of cartilage fragments or powder, and 30-60 weight percent of a synovial film.
在本發明之一些其他具體實施例中，所述組合物未事先與生物相容載體混合。所述組合物可直接施加至該軟骨缺陷中，並隨後將一適當載體或封閉劑施加至其中或其上以便能固定。較佳地，該載體或封閉劑係一纖維蛋白膠。In some other specific embodiments of the invention, the composition is not previously mixed with a biocompatible carrier. The composition can be applied directly to the cartilage defect, and a suitable carrier or sealant is then applied to or onto it to enable fixation. Preferably, the carrier or blocking agent is a fibrin glue.
本發明進一步由下列實例加以闡明，其係以例示之目的提供，而非限制之目的。The invention is further elucidated by the following examples, which are provided for illustrative purposes and not for restrictive purposes.
實例 1. 活體外研究 Example 1. In vitro studies
1. 骨髓濃縮物 (BMC) 之製備1. Preparation of Bone Marrow Concentrate (BMC)
豬的骨髓濃縮物係利用蔗糖梯度進行快速分離。簡言之，豬的骨髓係藉由注射器從脛骨抽取。骨髓隨後係以70微米篩孔濾過。加入至50毫升圓錐管中的17.5%蔗糖梯度 (15毫升) 中的經過濾之骨髓 (10毫升)，進行5分鐘的1,500 rpm離心一次。分餾層中的骨髓濃縮物隨後係透過進行5-10分鐘的1,500 rpm離心萃取出。將獲得的沉澱物分散至1毫升DPBS中，以便藉由自動細胞計數器來計算總細胞數。The pig's bone marrow concentrate was quickly separated using a sucrose gradient. In short, the bone marrow of a pig is drawn from the tibia by a syringe. The bone marrow was then filtered through a 70 micron sieve. Filtered bone marrow (10 ml) was added to a 17.5% sucrose gradient (15 ml) in a 50 ml conical tube and centrifuged once at 1,500 rpm for 5 minutes. The bone marrow concentrate in the fractionation layer was then extracted by centrifugation at 1,500 rpm for 5-10 minutes. The obtained pellet was dispersed into 1 ml of DPBS to calculate the total number of cells by an automatic cell counter.
2. 骨髓間質幹細胞 (BM-MSCs) 之製備2. Preparation of bone marrow mesenchymal stem cells (BM-MSCs)
從分餾層採集到的骨髓濃縮物係轉移至一新的試管中，隨後並以進行5-10分鐘的1,500 rpm離心，以獲得有核細胞。這些細胞係接種到培養燒瓶中並在含10% FBS的α-MEM中培養。The bone marrow concentrate collected from the fractionation layer was transferred to a new test tube, and then centrifuged at 1,500 rpm for 5-10 minutes to obtain nucleated cells. These cell lines were seeded into culture flasks and cultured in α-MEM containing 10% FBS.
3.軟骨組織碎片之製備3. Preparation of cartilage tissue fragments
軟骨組織從豬的膝關節獲取並以DPBS溶液清洗。以手術刀將組織剁碎成小碎片。尺寸大約1-2毫米 x 2-3毫米的軟骨碎片以DPBS清洗並進行5-10分鐘的1,500 rpm離心來收集。Cartilage tissue was obtained from the knee joints of pigs and washed with DPBS solution. Use a scalpel to chop the tissue into small pieces. Cartilage fragments of approximately 1-2 mm x 2-3 mm were washed in DPBS and centrifuged at 1,500 rpm for 5-10 minutes to collect.
4. 滑液膜碎片之製備4. Preparation of synovial membrane fragments
滑液膜從豬的膝關節獲取並以DPBS溶液清洗。以手術刀將組織剁碎成小碎片。尺寸大約2毫米 x 3毫米的滑液膜碎片以DPBS清洗並進行5-10分鐘的1,500 rpm離心來收集。Synovial membranes were obtained from the knee joints of pigs and washed with DPBS solution. Use a scalpel to chop the tissue into small pieces. Synovial membrane fragments of approximately 2 mm x 3 mm were washed in DPBS and centrifuged at 1,500 rpm for 5-10 minutes to collect.
5. 用於修復軟骨缺陷的組合物之製備5. Preparation of a composition for repairing cartilage defects
(1) BMC-軟骨碎片-滑液膜組合物(1) BMC-cartilage fragments-synovial membrane composition
將含有1-10 x 105 個細胞之骨髓濃縮物懸浮液及包含30-60毫克軟骨碎片及30-60毫克滑液膜之組織碎片混合在一起。離心之後，丟棄上層澄清液，並獲得BMC-軟骨碎片-滑液膜混合物。進一步 將該混合物在48孔培養盤上埋入於含有纖維蛋白原及凝血酶之0.2毫升TISSEEL溶液中以形成一生物構築體。所形成之構築體係轉移至24孔培養盤中並在含有10% FBS之α-MEM中培養。製備其他沒有組織碎片且沒有骨髓濃縮物的類似構築體供比較。Containing 1-10 x 10 5 cells of bone marrow suspension concentrates and tissue fragments comprising 30-60 mg fragments of cartilage and synovial membrane of 30-60 mg were mixed together. After centrifugation, the supernatant was discarded and a BMC-cartilage fragment-synovial membrane mixture was obtained. This mixture was further embedded in a 48-well culture plate in 0.2 ml of TISSEEL solution containing fibrinogen and thrombin to form a biological construct. The resulting building system was transferred to a 24-well culture plate and cultured in α-MEM containing 10% FBS. Other similar constructs were prepared without tissue debris and without bone marrow concentrate for comparison.
(2) BM-MSCs-軟骨碎片-滑液膜組合物 (供比較用)(2) BM-MSCs-cartilage fragments-synovial membrane composition (for comparison)
簡言之，BM-MSCs係經一系列的的胰蛋白酶作用。將含有1-10 x 104 個幹細胞之細胞懸浮液及包含30-60毫克軟骨碎片及30-60毫克滑液膜之組織碎片混合在一起。離心之後，丟棄上層澄清液，並獲得BM-MSCs-軟骨碎片-滑液膜混合物。進一步將該混合物在48孔培養盤上埋入於含有纖維蛋白原及凝血酶之0.2毫升TISSEEL溶液中以形成生物構築體。所形成之構築體係轉移至24孔培養盤中並在含有10% FBS之α-MEM中培養。製備其他具有不同BM-MSC數目的類似構築體供比較。In short, BM-MSCs undergo a series of trypsin actions. A cell suspension containing 1-10 x 10 4 stem cells and a tissue fragment containing 30-60 mg of cartilage debris and 30-60 mg of synovial membrane are mixed together. After centrifugation, the supernatant was discarded, and a BM-MSCs-cartilage fragment-synovial membrane mixture was obtained. This mixture was further embedded in a 48-well culture plate in 0.2 ml of TISSEEL solution containing fibrinogen and thrombin to form a biological construct. The resulting building system was transferred to a 24-well culture plate and cultured in α-MEM containing 10% FBS. Other similar constructs with different numbers of BM-MSC were prepared for comparison.
6. 基因表現上之RT-PCR分析6. RT-PCR analysis of gene expression
使用Trizol純化系統 (Invitrogen)依製造商的建議來分離全RNA。全RNA之反轉錄 (RT) 至單股cDNA係使用高容量cDNA RT套組 (Applied Biosystems)來完成。基因表現係使用ABI SteponePlusTM 即時PCR系統 (Applied Biosystems) 來分析。TaqMan® 探針係購自Applied Biosystems，其包括甘油醛-3-磷酸脫氫酶 (Hs99999905_m1)、軟骨可聚蛋白多糖 (Hs00153936_m1)、SOX9 (Hs00165814_m1)、第I型膠原蛋白 (Hs00164004_m1)、第II型膠原蛋白 (Hs00156568_m1)、及第X型膠原蛋白(Hs00166657_m1)。數據資料係估計為在構築體中細胞的mRNA量。週期閾值 (Ct) 係獲自每個樣本，並求取三次重複獲得之樣本值的平均。2- ΔΔ Ct 方法係用以計算每一目標基因的相對表現。Total RNA was isolated using the Trizol purification system (Invitrogen) according to the manufacturer's recommendations. Full RNA reverse transcription (RT) to single-stranded cDNA lines were performed using high-capacity cDNA RT kits (Applied Biosystems). Gene expression was analyzed using the ABI SteponePlus (TM) Instant PCR System (Applied Biosystems). TaqMan ® probes were purchased from Applied Biosystems and include glyceraldehyde-3-phosphate dehydrogenase (Hs99999905_m1), cartilage polymerizable proteoglycan (Hs00153936_m1), SOX9 (Hs00165814_m1), type I collagen (Hs00164004_m1), type II Type collagen (Hs00156568_m1) and type X collagen (Hs00166657_m1). The data is estimated as the amount of mRNA in the cells in the construct. The cycle threshold (Ct) is obtained from each sample and the average of the sample values obtained from three replicates is calculated. The 2 - ΔΔ Ct method is used to calculate the relative performance of each target gene.
7. 結果7. Results
以下組別之培養物在第II型膠原蛋白之基因表現上係經RT-PCR分析，且結果係顯示於圖式中：(A1) BMC (5 x 105 ) (圖 1A )；(A2) 軟骨-滑液膜 (圖1B )；(A3) BMC (5 x105 )-軟骨-滑液膜 (圖 1C )；(B1) BM-MSCs (5 x 105 )-軟骨-滑液膜 (圖 1F )；以及 (B2) BM-MSCs (5 x 104 )-軟骨-滑液膜 (圖 1G )。圖 1D 顯示組別A3之結果相對於組別A1之結果的比較，以及圖 1E 顯示組別A3之結果相對於組別A2之結果的比較。The following groups were cultured in the gene expression of type II collagen-based proteins by RT-PCR analysis, and the results are shown in line drawings: (A1) BMC (5 x 10 5) ( FIG. 1A); (A2) cartilage - synovial membrane (FIG. 1B); (A3) BMC ( 5 x10 5) - cartilage - synovial membrane (FIG. 1C); (B1) BM- MSCs (5 x 10 5) - cartilage - synovial membrane (FIG. 1F); and (B2) BM-MSCs (5 x 10 4) - cartilage - synovial membrane (FIG. 1G). FIG. 1D shows a comparison of the result of group A3 with respect to the result of group A1, and FIG. 1E shows a comparison of the result of group A3 with respect to the result of group A2.
組別A3 (根據本發明之組合物) 相較於組別A1 (只有BMC) 及A2 (只有軟骨-滑液膜)，顯示第II型膠原蛋白表現隨培養時間有較多的增加 (直到6個星期) (參見圖 1A-1E )。同時在使用BM-MSCs而非BMC之組別B1及B2的情況中，第II型膠原蛋白表現則並未隨培養時間而有顯著增加。Group A3 (composition according to the invention) showed a greater increase in type II collagen performance with culture time compared to groups A1 (only BMC) and A2 (only cartilage-synovial membrane) (up to 6 Weeks) (see Figures 1A-1E ). At the same time, in the case of using BM-MSCs instead of BMC in groups B1 and B2, the performance of type II collagen did not increase significantly with the culture time.
第II型膠原蛋白在關節軟骨 (或透明軟骨) 之細胞外基質 (ECM) 中的膠原蛋白中佔90%至95%，並因此被認為對成功的軟骨缺陷修復中是關鍵性的。Type II collagen accounts for 90% to 95% of the collagen in the extracellular matrix (ECM) of articular cartilage (or hyaline cartilage) and is therefore considered critical for successful cartilage defect repair.
實例 2. 動物研究 Example 2. Animal research
1. 骨髓濃縮物 (BMC) 之製備1. Preparation of Bone Marrow Concentrate (BMC)
豬的骨髓濃縮物係利用蔗糖梯度進行快速分離。簡言之，10毫升豬的骨髓係藉由注射器從脛骨抽取。加入至50毫升圓錐管中的17.5%蔗糖梯度 (15毫升) 中的經過濾之骨髓 (10毫升)，進行5分鐘的1,500 rpm離心一次。分餾層中的骨髓濃縮物隨後係透過5-10分鐘的1,500 rpm離心萃取出。沉澱物係分散至1毫升DPBS中，以便藉由自動細胞計數器來計算總細胞數。The pig's bone marrow concentrate was quickly separated using a sucrose gradient. Briefly, the bone marrow of a 10 ml pig was drawn from the tibia by a syringe. Filtered bone marrow (10 ml) was added to a 17.5% sucrose gradient (15 ml) in a 50 ml conical tube and centrifuged once at 1,500 rpm for 5 minutes. The bone marrow concentrate in the fractionation layer was then extracted by centrifugation at 1,500 rpm for 5-10 minutes. The pellet was dispersed in 1 ml of DPBS to calculate the total number of cells by an automatic cell counter.
2. 軟骨組織碎片之製備2. Preparation of cartilage tissue fragments
軟骨組織從豬的膝關節獲取並以DPBS溶液清洗。以手術刀將組織剁碎成小碎片。尺寸大約1-2毫米 x 2-3毫米的軟骨碎片以DPBS清洗並進行5-10分鐘的1,500 rpm離心來收集。Cartilage tissue was obtained from the knee joints of pigs and washed with DPBS solution. Use a scalpel to chop the tissue into small pieces. Cartilage fragments of approximately 1-2 mm x 2-3 mm were washed in DPBS and centrifuged at 1,500 rpm for 5-10 minutes to collect.
3. 滑液膜碎片之製備3. Preparation of synovial membrane fragments
滑液膜從豬的膝關節獲取並以DPBS溶液清洗。以手術刀將組織剁碎成小碎片。尺寸大約2毫米 x 3毫米的滑液膜碎片以DPBS清洗並進行5-10分鐘的1,500 rpm離心來收集。Synovial membranes were obtained from the knee joints of pigs and washed with DPBS solution. Use a scalpel to chop the tissue into small pieces. Synovial membrane fragments of approximately 2 mm x 3 mm were washed in DPBS and centrifuged at 1,500 rpm for 5-10 minutes to collect.
4. 活體內動物模型Animal model in vivo
在本研究中所採用的是小型豬。所有的豬隻在進行手術時都已經性成熟。所有的操作及介入均在全身麻醉下執行。在兩膝關節之內側髁中製成全厚度的缺陷 (參見圖 2A )，其中兩者之一接受包含骨髓濃縮物、60毫克軟骨碎片及60毫克滑液膜之組合物，以及隨後的Tisseel溶液 (纖維蛋白膠) (0.2-0.4 毫升) 以固定；以及另一者接受微斷裂處理以作為對照組。缺陷的直徑為8毫米且深度為1毫米。Mini pigs were used in this study. All pigs were sexually mature at the time of surgery. All procedures and interventions were performed under general anesthesia. A full-thickness defect was made in the medial condyle of both knees (see Figure 2A ), one of which received a composition containing bone marrow concentrate, 60 mg of cartilage fragments and 60 mg of synovial membrane, and subsequent Tisseel solution (Fibrin glue) (0.2-0.4 ml) to be fixed; and the other received a micro-fracture treatment as a control group. The defect has a diameter of 8 mm and a depth of 1 mm.
5. 結果5. Results
觀察到的是，在施加至組合物上之後，纖維蛋白膠表現高度黏合性，且纖維蛋白膠以及組合物可保持在缺陷位置處，而不會四處流散，儘管從缺陷位置處有出血的情況，並因此不需要骨膜移植或骨膜縫合以便使組合物固定在位置上。相反地，已知的組合物會從缺陷位置處流走，並需要在手術時進行骨膜縫合 (或其他膜縫合)。參見圖 2B 及2C 。It was observed that after application to the composition, the fibrin glue exhibited a high degree of adhesion, and the fibrin glue and the composition could remain at the defect site without being scattered around, despite bleeding from the defect site And therefore does not require a periosteal graft or periosteal suture to hold the composition in place. In contrast, known compositions flow away from the defect site and require periosteal suture (or other membrane suture) during surgery. See Figures 2B and 2C .
在從手術復原的階段裡，允許動物自由活動。手術之後，豬隻行走緩慢且不會久站，除了進食的時候之外。牠們趴在地板上休息。這樣類型的行為持續了幾天，而在這段時間過後豬隻們的行為便回歸往常。移植後六個月觀察到良好的軟骨缺陷修復 (圖 2D )。Animals are allowed to move freely during the recovery phase from surgery. After the operation, the pigs walk slowly and will not stand for long, except when eating. They lie on the floor and rest. This type of behavior lasted for a few days, after which the pigs' behavior returned to normal. Good repair of cartilage defects was observed six months after transplantation ( Figure 2D ).
所屬技術領域的技藝人士將會明瞭，不背離主要的發明概念下可對上述的具體實施例進行修改。因此，可以理解的是，本發明並不限於所揭示的特定具體實施例，而是意欲涵蓋申請專利範圍所定義之本發明精神與範圍內的任何修飾。Those skilled in the art will appreciate that the specific embodiments described above may be modified without departing from the main inventive concept. Therefore, it can be understood that the present invention is not limited to the specific embodiments disclosed, but is intended to cover any modification within the spirit and scope of the present invention as defined by the scope of the patent application.
無no
配合隨附圖式閱讀將可較佳地理解前述之發明內容、以及下方的本發明實施方式。為說明本發明，圖式中所示之具體實施例中為目前較佳的。The foregoing summary of the invention and the embodiments of the invention below can be better understood by reading the accompanying drawings. To illustrate the present invention, the specific embodiments shown in the drawings are currently preferred.
在圖式中：In the scheme:
圖1A顯示組別A1 (BMC (5 x 105))之培養的RT-PCR結果。Figure 1A shows the results of RT-PCR of the culture of group A1 (BMC (5 x 105)).
圖1B顯示組別A2 (軟骨-滑液膜)之培養的RT-PCR結果。***p ＜ 0.001。FIG. 1B shows the results of RT-PCR of culture of group A2 (cartilage-synovial membrane). *** p <0.001.
圖1C顯示組別A3 (BMC (5 x105)- 軟骨-滑液膜)之培養的RT-PCR結果。***p ＜ 0.001。Figure 1C shows the results of RT-PCR of the culture of group A3 (BMC (5 x 105)-cartilage-synovial membrane). *** p <0.001.
圖1D顯示組別A3之結果對組別A1之結果的比較。FIG. 1D shows a comparison of the results of group A3 to the results of group A1.
圖1E顯示組別A3之結果對組別A2之結果的比較。FIG. 1E shows a comparison of the results of group A3 to the results of group A2.
圖1F顯示組別B1 (BMC-MSCs (5 x105)- 軟骨-滑液膜)之培養的RT-PCR結果。Figure 1F shows the results of RT-PCR of the culture of group B1 (BMC-MSCs (5 x 105)-cartilage-synovial membrane).
圖1G顯示組別B2 (BMC-MSCs (5 x104)- 軟骨-滑液膜)之培養的RT-PCR結果。Figure 1G shows the results of RT-PCR of the culture of group B2 (BMC-MSCs (5 x104)-cartilage-synovial membrane).
圖2A係顯示一產生之軟骨缺陷的照片。Figure 2A is a photograph showing a cartilage defect.
圖2B係顯示本發明之組合物可保持在缺陷位置處的照片。FIG. 2B is a photograph showing that the composition of the present invention can be held at a defect position.
圖2C 係顯示在使用本發明用於軟骨缺陷修復之組合物時不需要骨膜移植或骨膜縫合的照片。FIG. 2C is a photograph showing that a periosteum transplantation or a periosteum suture is not required when using the composition for cartilage defect repair of the present invention.
圖2D係顯示移植後6個月之軟骨修復的照片。Figure 2D is a photograph showing cartilage repair 6 months after transplantation.
無no
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| WO2015048317A1 (en) * | 2013-09-25 | 2015-04-02 | The Children's Mercy Hospital | Decellularized hyaline cartilage powder for tissue scaffolds |
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