Composite grafts of demineralized compact bone and marrow under systemic biosynthetic growth hormone treatment

The influence of systemic treatment with biosynthetic human growth hormone on bone formation was studied, comparing rabbit radial bone defects grafted with demineralized autologous bone with contrallateral defects grafted with a composite including bone marrow. At 14 and 28 days after operation the bone-forming process was evaluated by roentgenography (planimetry included), and by scintigraphy after injection of 99mTc-labeled dicarboxypropane diphosphonate. At 28 days autoradiography was also performed. The roentgenographic and scintigraphic examinations revealed no difference between the grafted sides at either 14 or 28 days postoperatively. The elimination, after 14 days' growth hormone treatment, of a previously found difference between the marrow- and the nonmarrow-grafted sides might be an indication of accelerated osteogenesis at the beginning of the healing process.     

Composite grafts of demineralized compact bone and marrow under systemic biosynthetic growth hormone treatment

Summary The influence of systemic treatment with biosynthetic human growth hormone on bone formation was studied, comparing rabbit radial bone defects grafted with demineralized autologous bone with contrallateral defects grafted with a composite including bone marrow. At 14 and 28 days after operation the bone-forming process was evaluated by roentgenography (planimetry included), and by scintigraphy after injection of ^99mTc-labeled dicarboxypropane diphosphonate. At 28 days autoradiography was also performed. The roentgenographic and scintigraphic examinations revealed no difference between the grafted sides at either 14 or 28 days postoperatively. The elimination, after 14 days' growth hormone treatment, of a previously found difference between the marrow- and the nonmarrow-grafted sides might be an indication of accelerated osteogenesis at the beginning of the healing process.     

Bridging large defects in bone by demineralized bone matrix in the form of a powder. A radiographic, histological, and radioisotope-uptake study in rats

Demineralized bone powder was used as an osteoinductive substance to bridge very large defects (more than 50 per cent of the total length of the bone) in one radius of each of thirty-three rats. An identical defect was produced in the contralateral radius of each animal for use as a control. The defect on the control side was left unbridged or was bridged by large chips of autologous bone or an autologous inlay graft. All rats showed formation of new bone throughout the length of the radial defect only on the side in which the demineralized bone powder had been implanted. The control side, in which an autologous graft in the form of chips or inlay had been implanted, showed resorption of the graft. The maximum rate of formation of bone occurred fifteen to twenty-one days after implantation of the demineralized bone powder. At thirty-five days, the experimental defect was fully bridged, forming solid bone, in 71 per cent of the rats, and the remaining 29 per cent showed bridging of 95.8 per cent of the length of the defect, with union on one side. Analysis of the sequential radiographs, technetium-99m scans, and histological findings showed that the formation of bone and bridging of the defect were superior on the side in which the demineralized bone powder had been implanted compared with the side in which pieces of autologous bone or an autologous inlay graft had been used.(ABSTRACT TRUNCATED AT 250 WORDS)     

外源性重组人骨形态发生蛋白-2应用于兔腰椎植骨融合中细胞增殖的实验研究

目的 探讨外源性重组人骨形态发生蛋白-2(rhBMP-2)应用于兔腰椎后路横突植骨融合中的促成骨效应细胞增殖作用及其成骨机制. 方法 45只新西兰大白兔随机分为三组(n=15),建立腰椎后路横突间植骨融合模型,分别植入rhBMP-2/异体骨复合骨条(复合骨组)、自体髂骨条(自体骨组)、单纯异体髂骨条(异体骨组).用流式细胞仪检测2、7、14、28、35 d具有成骨效应的骨髓基质细胞(MSCs)、成骨细胞、血管内皮细胞的增殖量. 结果复合骨组MSCs增殖量在术后2、7、35 d均比自体骨组和异体骨组高,差异均有统计学意义(P<0.05).复合骨组成骨细胞增殖量除在术后2 d高于自体骨组,差异有统计学意义(P=0.028)外,在其他时间点差异均无统计学意义(P>0.05),但复合骨组成骨细胞增殖量在术后2、7、14、28、35 d时均高于异体骨组,差异有统计学意义(P<0.05).复合骨组血管内皮细胞增殖量在术后2、7、28 d均高于自体骨组和异体骨组,差异有统计学意义(P<0.05).结论 在脊柱融合的不同时间段,外源性rhBMP-2能有效地促进MSCs、成骨细胞、血管内皮细胞增殖.     

Bone formation in demineralized bone transplants treated with biosynthetic human growth hormone

The influence of local administration of biosynthetic human growth hormone on the bone formation in a rabbit bone defect grafted with demineralized autologous bone was studied and compared to contralateral saline administration. The growth hormone was produced by hybrid DNA-technique and administered locally to the bone defect with the demineralized bone implant by an osmotic pump delivering 5 microliters/h during 14 days. At 14 and 28 days after operation the bone forming process was evaluated by roentgenography, planimetry included, and scintigraphy after injection of 99Tcm-labelled DPD. At 28 days after operation also autoradiography was performed. The roentgenographic and scintigraphic examinations revealed no influence from growth hormone neither 14 nor 28 days postoperatively. Centrally in the remineralized grafts the roentgenograms 28 days after operation showed radiolucent areas which in corresponding autoradiograms showed no uptake of 99Tcm. These areas turned out to be remnants of matrix. It is suggested that growth hormone has no influence on the bone induction process during the first 14 days when administered to bone matrix, probably due to relative lack of osteogenic cells. This study contributes a base for further evaluation of factors pertinent to hormonal stimulation of bone matrix transplants.     

The use of demineralized bone matrix in the repair of segmental defects. Augmentation with extracted matrix proteins and a comparison with autologous grafts

A soluble protein component of bone, bone morphogenetic protein, and decalcified bone matrix have been shown to induce the formation of bone in extraosseous tissue. Clinical and animal studies investigating the use of these materials as bone grafts have shown radiographic and histological evidence of formation of bone, but the clinical usefulness of these grafts remains unknown. This study compared the healing processes when plasma-coated demineralized bone matrix and autologous cancellous bone were used to graft segmental defects of bone. A standard procedure was used to make a two-centimeter defect bilaterally in the ulna of forty-eight skeletally mature New Zealand White rabbits. In each rabbit, one ulnar defect was grafted with autologous citrated plasma-coated demineralized bone matrix while the other defect served as a control and was grafted with either autologous cancellous bone from the iliac crest, demineralized bone matrix, or demineralized bone matrix augmented with bone proteins that had been extracted with guanidinium hydrochloride. The ulnar defect was stabilized by the intact radius, and no supplemental device was necessary for fixation. To examine spontaneous healing in this model, one group of rabbits had a control defect that was not grafted. The grafts were periodically evaluated by radiographs, and twelve weeks after surgery the grafts were harvested and tested to failure in a standard torsion-test machine. The mechanical parameters were calculated, and histological examination of major fragments of the grafts was performed. The results of the radiographic and histological evaluation showed that all of the grafted ulnae healed, with fusion of the graft to the cut ends of the defect and reformation of approximately normal anatomy. No ungrafted ulnar defects healed. The results from the mechanical tests were evaluated by comparing the defect that was grafted with plasma-coated demineralized bone matrix with the control graft in each animal. These data showed that: twelve weeks after grafting, the normal ulnae were significantly stronger than the ulnae that had been grafted with plasma-coated demineralized bone matrix; the ulnae that had been grafted with plasma-coated demineralized bone matrix and those that had been grafted with autologous bone were equivalent in strength; and twelve weeks after grafting, grafts of demineralized bone matrix that were augmented with extracted bone proteins were significantly stronger than those that had not been so augmented.     

骨形态发生蛋白复合物联合自体骨髓移植治疗四肢长管状骨骨不连

目的:观察骨形态发生蛋白复合物联合自体红骨髓移植治疗四肢长管状骨骨不连的临床疗效.方法:自2004年1月至2010年12月,采用骨形态发生蛋白复合物联合自体红骨髓移植法治疗四肢长管状骨骨不连患者36例,男22例,女14例;年龄22～68岁,平均36.8岁.骨不连部位:肱骨6例,尺骨4例,桡骨3例,股骨10例,胫骨13例.伤后距治疗时间8～24个月,平均13.7个月.增生性骨不连22例,萎缩性骨不连14例 患者主要临床症状为骨折部位负重时疼痛,肢体肿胀X线片提示骨折线清晰伴骨缺损.术后通过观察手术切口愈合、植骨区及骨髓穿刺区反应、骨折愈合、邻近的关节功能恢复等情况评估手术疗效.结果:术后患者切口均甲级愈合,未发现过敏和免疫排斥反应.骨髓穿刺区无感染及血肿形成.36例患者均获随访,时间3～28个月,平均16.2个月.无骨髓穿刺区慢性疼痛、植骨区骨质感染、切口周围皮肤红肿或窦道形成.骨不连均获得骨性愈合,愈合时间3～12个月,平均6.2个月,无畸形愈合.骨不连愈合后骨折部位负重时疼痛消失、肢体肿胀消退.5例患者遗留邻近关节功能部分受限,其余患者均完全恢复.结论:骨形态发生蛋白复合物联合自体红骨髓移植在治疗四肢长管状骨骨不连中具有来源广泛、安全可靠、加速骨愈合等优点,是其理想的植骨材料之一.     

Osteogenic effect of a gastric pentadecapeptide, BPC-157, on the healing of segmental bone defect in rabbits: a comparison with bone marrow and autologous cortical bone implantation

Gastrectomy often results in increased likelihood of osteoporosis, metabolic aberration, and risk of fracture, and there is a need for a gastric peptide with osteogenic activity. A novel stomach pentadecapeptide, BPC-157, improves wound and fracture healing in rats in addition to having an angiogenic effect. Therefore, in the present study, using a segmental osteoperiosteal bone defect (0.8 cm, in the middle of the left radius) that remained incompletely healed in all control rabbits for 6 weeks (assessed in 2 week intervals), pentadecapeptide BPC-157 was further studied (either percutaneously given locally [10 microg/kg body weight] into the bone defect, or applied intramuscularly [intermittently, at postoperative days 7, 9, 14, and 16 at 10 microg/kg body weight] or continuously [once per day, postoperative days 7-21 at 10 microg or 10 ng/kg body weight]). For comparison, rabbits percutaneously received locally autologous bone marrow (2 mL, postoperative day 7). As standard treatment, immediately after its formation, the bone defect was filled with an autologous cortical graft. Saline-treated (2 mL intramuscularly [i.m.] and 2 mL locally into the bone defect), injured animals were used as controls. Pentadecapeptide BPC-157 significantly improved the healing of segmental bone defects. For instance, upon radiographic assessment, the callus surface, microphotodensitometry, quantitative histomorphometry (10 microg/kg body weight i.m. for 14 days), or quantitative histomorphometry (10 ng/kg body weight i.m. for 14 days) the effect of pentadecapeptide BPC-157 was shown to correspond to improvement after local application of bone marrow or autologous cortical graft. Moreover, a comparison of the number of animals with unhealed defects (all controls) or healed defects (complete bony continuity across the defect site) showed that besides pentadecapeptide intramuscular application for 14 days (i.e., local application of bone marrow or autologous cortical graft), also following other pentadecapeptide BPC-157 regimens (local application, or intermittent intramuscular administration), the number of animals with healed defect was increased. Hopefully, in the light of the suggested stomach significance for bone homeostasis, the possible relevance of this pentadecapeptide BPC-157 effect (local or intramuscular effectiveness, lack of unwanted effects) could be a basis for methods of choice in the future management of healing impairment in humans, and requires further investigation.     

自体松质骨加骨髓移植治疗陈旧性腕舟骨骨折不连的临床研究

目的观察自体松质骨加骨髓移植治疗陈旧性腕舟骨骨折不连及近端坏死型腕舟骨骨折的临床效果。方法随机选择14例陈旧性腕舟骨骨折不连患者，搔刮舟骨远、近两端死骨，保留皮质骨壳，取自体松质骨（桡骨远端或髂骨）移植至舟骨远、近两端，舟骨复位后交叉克氏针固定。髂骨内抽取自体红骨髓5ml，快速、加压注入舟骨骨折部位。术后6周开始，每周拍摄计算机X线片（CR-X）一次，至骨折愈合，并记录骨折愈合及恢复工作时间。结果术后随访10周～5年，14例陈旧性腕舟骨全部愈合，骨折愈合时间为8～12周，平均9．3周。13例腕关节活动度达到健侧腕部标准，活动时无疼痛，恢复了原来工作；1例较术前有改善，但腕关节活动未达健侧标准，且活动时疼痛。结论自体松质骨加自体骨髓移植治疗陈旧性腕舟骨骨折不连，较传统治疗方法，缩短了骨折愈合时间，提高了治愈率，保留了原解剖结构和生物力学特性，是一种有效的治疗方法。     

人工骨联合自体骨髓移植技术的骨缺损修复研究与应用

目的分析人工骨联合自体骨髓移植技术治疗骨缺损的临床疗效。方法选取2011年4月至2013年9月,来本院诊治的四肢粉碎性骨折术后骨缺损患者40例,随机分为两组（A、B组）,分别行自体髂骨植骨和人工骨联合自体骨髓移植治疗四肢粉碎性骨折所致骨缺损。术后观察两组骨折愈合时间,骨折愈合率,并发症的发生及骨缺损的修复和功能重建优良率。结果两组均行6～12个月随访,平均（9.14±1.36）月,所有患者植骨术后均无切口感染、发热等并发症。A组15例骨缺损区愈合良好,住院时间平均（19.36±2.54）天,骨折愈合时间平均（5.67±1.52）个月,骨缺损的修复和功能重建评价标准,优12例,良3例,可3例,差2例,优良率75%;B组18例骨缺损区愈合良好,住院时间平均（12.19±1.52）天,骨折愈合时间平均（3.61±1.13）个月,骨缺损的修复和功能重建评价标准,优16例,良2例,可2例,差0例,优良率90%。B组骨缺损治疗效果显著优于A组。结论人工骨联合自体骨髓移植较单纯自体髂骨移植治疗骨缺损更能促进骨痂生长,加速骨折后骨缺损愈合,更加有效地减少住院时间及骨折愈合时间,骨折愈合率更高,骨缺损修复和功能重建效果更加显著。     

The effect of a composite of polyorthoester and demineralized bone on the healing of large segmental defects of the radius in rats.

The effect of a composite of demineralized bone mixed with polyorthoester on the healing of large segmental defects in the rat radius was studied. Sixty male Wistar rats were divided into four groups, A through D, and an osteoperiosteal diaphyseal defect of 50 per cent of the length of the bone was made in the right radius of each rat. In Group A, the defect was filled with polyorthoester and demineralized bone; in Group B, demineralized bone; and in Group C, polyorthoester. No material was implanted in the defects in the Group-D rats. The rats were killed fifty days postoperatively. The formation of bone in the defects was quantified with computer-assisted measurements of the area on radiographs. The host-tissue response was evaluated with light microscopy. Defects that had been filled with the composite of polyorthoester and demineralized bone or with demineralized bone alone showed regeneration of bone corresponding to 93.6 and 77.6 per cent of the area of the defect, respectively. Defects that had no implant or that had been filled with polyorthoester alone showed significantly less formation of bone. No inflammation was seen with light microscopy, and only traces of the polyorthoester could be detected in the defects that had been filled with the composite or with polyorthoester alone.     

Bone matrix and marrow versus cancellous bone in rabbit radial defects

Implants of demineralized bone matrix induce new bone formation. In order to estimate the possible clinical usefulness of this phenomenon, autologous cancellous bone grafts were compared with composite grafts of bone matrix and marrow. Cancellous bone from the tuber ischii of the rabbit was transplanted to a preformed radial defect in the same animal. On the opposite side, a similar defect was filled with a mixture of either allogenous or autogenous bone-matrix particles and autogenous bone marrow. After 25 days, calcium 45 was injected intravenously. Three days later the animals were killed. Standardized segments of the rabbit's forearms, containing the middle of the defect, were cut out, ashed, and analyzed for 45Ca activity. No side difference in 45Ca deposition was found. The callus ash weight of the allogenous matrix-transplanted side was approximately 60% of that of the cancellous bone side. This side difference of ash weights corresponds to the estimated initial mineral content of the cancellous graft. Nontransplanted defects had very low ash weight and 45Ca activity. Thus, in the rabbit, composite grafts of bone matrix and marrow produce a bone yield comparable to that of cancellous bone.     

Osseous healing with a composite of allograft and demineralized bone matrix: adverse effects of smoking

We report on our use of a composite graft of lyophilized cancellous allogenic chips and demineralized bone matrix (DBM; Grafton; Osteotech, Eatontown, NJ) to manage traumatic osseous defects and nonunions. Data were prospectively collected from all patients who received this composite bone graft between 1996 and 2000. Only acute fractures with bone loss resulting in a uncontained defect and atrophic non-unions were included in the present study. Demographic data and complications related to composite use, tobacco use, and other comorbidities that could affect healing were evaluated. One hundred seven patients (112 bone graft sites) were followed up for a mean of 32 months (range, 12-60 months). Graft sites included the forearm, femur and tibia. Of the 112 patients, there were 56 smokers (25 non-unions and 31 fractures) and 56 non-smokers (28 fractures and 28 non-unions). Healing occured in 38/56 smokers compared with 49/56 non-smokers. In failed cases, smoking was characteristic in 7/9 non-unions and 11/16 fractures. There were 26 acute uncontained injuries, 29 acute contained defects, and 67 nonunions. Grafting sites were radius/ulna (13 cases), humerus (17), femur (31), and tibia/fibula (51). Significant comorbidities were diabetes mellitus (4 cases), fungal osteomyelitis (1), and pulmonary alveolar proteinosis (1). Eight (73%) of the 11 patients with graft failure had a significant smoking history. This composite graft is an option for managing osseous defects and nonunions traditionally treated with autologous bone grafting but should be used with caution when treating patients who are smokers.     

Bone matrix and marrow versus cancellous bone in rabbit radial defects

Summary Implants of demineralized bone matrix induce new bone formation. In order to estimate the possible clinical usefulness of this phenomenon, autologous cancellous bone grafts were compared with composite grafts of bone matrix and marrow. Cancellous bone from the tuber ischii of the rabbit was transplanted to a preformed radial defect in the same animal. On the opposite side, a similar defect was filled with a mixture of either allogenous or autogenous bone-matrix particles and autogenous bone marrow. After 25 days, calcium 45 was injected intravenously. Three days later the animals were killed. Standardized segments of the rabbit's forearms, containing the middle of the defect, were cut out, ashed, and analyzed for ⁴⁵Ca activity. No side difference in ⁴⁵Ca deposition was found. The callus ash weight of the allogenous matrix-transplanted side was approximately 60% of that of the cancellous bone side. This side difference of ash weights corresponds to the estimated initial mineral content of the cancellous graft. Nontransplanted defects had very low ash weight and ⁴⁵Ca activity. Thus, in the rabbit, composite grafts of bone matrix and marrow produce a bone yield comparable to that of cancellous bone.     

自体深筋膜复合自体红骨髓移植治疗骨不连

目的：根据膜引导性骨再生与骨诱导再生的理论,将自体深筋膜复合自体红骨髓移植治疗骨不连,为临床寻找一种简便有效的治疗骨不连的方法。方法：自2006年3月至2009年3月,采用自体深筋膜复合自体红骨髓移植的方法对17例骨不连患者进行治疗,男10例,女7例;年龄7～52岁,平均32岁;胫骨10例,桡骨5例,锁骨2例。受伤至入院时间7～36个月,平均12个月。入院前已手术1次者10例,2次者5例,3次者2例。骨不连部位均位于骨干,皮肤软组织条件较好。X线片显示增生性骨不连11例,萎缩性骨不连6例。均拆除原有内固定物,重新用髓内钉或钢板内固定,并用自体深筋膜密闭缝合骨折端,内注入自体红骨髓。结果：17例患者均获得随访,时间5个月～2年,平均1年。骨折临床愈合时间12～20周,平均16周。术后根据骨折愈合标准对疗效进行评估：优14例,良2例,差1例。结论：自体深筋膜复合自体红骨髓移植治疗骨不连,骨折愈合时间短,适合于骨不连位于骨干,皮肤软组织条件较好的患者。     

四肢难治性骨不连的三种治疗策略疗效分析

[目的] 比较分析3种方法治疗四肢难治性骨不连的临床疗效.[方法] 2003年4月-2008年2月,共收治四肢难治性骨不连36例39肢,男22例,女14例;年龄15～71岁,平均51.5岁;胫骨19肢,肱骨9肢,股骨7肢,尺、桡骨4肢;治疗骨不连手术次数1～4次,平均2.5次;术前X线片骨不连骨断端距离:4.5～36 mm,平均16.5 mm;下肢短缩距离:5～40 mm,平均21 mm.自体骨髓复合人工骨联合髂骨骨膜移植19肢(联合组),自体骨髓移植联合人工骨9肢(骨髓组),自体髂骨移植治疗11肢(髂骨组).[结果] 39肢最终均达骨性愈合,均获随访12～28个月,平均18.5个月.骨愈合时间、固定物取出1个月患肢功能评分和术后X线片评分,联合组疗效优越(P＜0.05).联合组骨愈合时间(5.5±1.5)个月,骨髓组骨愈合时间(6.6±1.8)个月,髂骨组骨愈合时间(7.4±2.1)个月.[结论] 自体骨髓复合人工骨联合髂骨骨膜移植治疗四肢难治性骨不连临床疗效优越.     

骨内膜成骨的动物模型

目的 为了解骨内膜在骨折愈合不同阶段的功能变化及其调节机制。设计一个骨折愈合过程中骨内膜成骨的动物模型。方法 将３月龄雌性ＳＤ大鼠４０只随机分成４组,每组１０只,在左胫骨造成长５ｍｍ的环形骨膜缺损,用骨锉在骨膜缺损区造成长５ｍｍ的骨缺损,深达髓腔,约显露髓腔的１／６。结果 １４天时裸露的贿腔已开始出现骨性关闭的征象,２８天时髓腔完全由坚质骨关闭,而各时间点组的骨缺损处填充物均为颜痕样结缔组织。结论     

固骼生联合自体髂骨在发育性髋关节脱位患儿行髋臼截骨术中的应用

 目的 比较单纯应用自体髂骨植骨与联合应用固骼生植骨对发育性髋关节脱位(developmental dislocation of the hip, DDH)患儿行髋臼截骨术的临床效果。方法 回顾性分析2007年3月至2011年10月采用切开复位髋臼截骨术治疗的113例DDH患儿资料。根据髋臼截骨术后使用的植骨材料,分为自体髂骨植骨组(52例,60髋)与固骼生联合自体髂骨植骨组(61例,67髋)。113例患儿髋关节Tonnis脱位分级均为Ⅱ～Ⅳ度。两组患儿性别、年龄、手术侧别、脱位分型、髋臼截骨方式及髋臼指数比较,差异均无统计学意义。分别于术后6周、3个月、6个月、1年与2年进行随访,分别比较两组患儿髋臼截骨区骨愈合情况、髋关节的影像学及功能恢复情况,并采用Lane、Severin 及McKay标准进行评分。结果 术后6周与术后3个月时Lane骨愈合评分,联合固骼生组分别为(6.4±1.3)分、(9.6±1.7)分,自体髂骨组分别为(4.7±1.5)分、(7.8±1.2)分,前者骨愈合情况明显优于后者,两组之间有差异;而术后6个月与术后1年时,联合固骼生组和自体髂骨组截骨区均基本达到骨性愈合,两组无差异。Severin标准髋关节影像学评价结果显示：联合固骼生组(优41髋、良22髋、可4髋)优良率为(94.0%,63/67)明显高于自体髂骨组(优28髋、良22髋、可10髋)的优良率(83.3%,50/60);自体髂骨组可的概率(16.7%,10/60)明显高于联合固骼生组(6.0%,4/67)。McKay标准髋关节功能评价结果与影像学评价结果一致。结论 固骼生作为一种新型的骨缺损修复材料,能在术后早期促进髋臼截骨区骨愈合,从而提高截骨区域抗压性。在DDH患儿行髋臼截骨术中联合应用固骼生植骨可以辅助获得满意的头臼对位关系及髋关节功能。     

Implants of heterologous demineralized bone matrix for induction of posterior spinal fusion in rats.

The authors tested the osteoinductive capacity of powdered heterologous (bovine) demineralized bone matrix in rats. The first part of the study concerned a monolateral posterior spinal implant after decortication of three vertebrae, using as a control area the animal's contralateral side, in which neither bone graft nor any other material were placed. In another group of rats, a comparative evaluation was made of powdered heterologous demineralized bone matrix and fresh autologous bone. In the same animal, autologous bone was implanted to realize a thoracic posterior fusion and demineralized bone matrix, to induce a posterior fusion in the lumbar area. All data obtained suggested a good osteoinductive activity of heterologous powdered demineralized bone matrix. The two posterior spinal fusions done in the same animal with heterologous demineralized bone matrix or autologous bone, respectively, had similar callus development and required the same time for formation.