il技术肢体延长生物学基础

1、北京骨外固定技术研究所 市场专员 成江涛 Dr. Gang Li BMed, D.Phil (Oxon), Senior LecturerDept of Orthopaedic Surgery, Queens University Belfast, Musgrave Park Hospital, Belfast, N. Ireland, UKBasic Concepts of Bone Formation During Fracture Healing and Distraction Osteogenesis北京骨外固定技术研究所 市场专员 成江涛 Distraction osteogenes

2、is Prof. G.A. Ilizarov Codivilla, Alessandro(Am. J. Orthop. Surg 1905)Ilizarov, G.A. - Tensition-Stress PrincipleClin Orthop, 238 & 239, 1989Experimental and Clinical studiesDOG: Ilizarov; Aronson, Delloye, etc. RABBIT: Kawamura (1968), Kojimoto, Hamanishi, Kenwright, Simpson, Li and othersSHEEP

3、, RAT and MOUSE: Yasui, Karaharju, Ippulito,Rat Aronson, Helms, etc.HUMAN: Kawamura, Ilizarov, Paley, Hamanishi, Simpson , etc.北京骨外固定技术研究所 市场专员 成江涛 Summary of previous findings:Understanding of the surgical techniques: age of patientfixator selection corticotomy or osteotomylatency period 5-10perios

4、teum or soft tissuelengthening rate: 0.5-0.7 mm/dayrhythm: twice or more per day flexibility and physiotherapyExperience 北京骨外固定技术研究所 市场专员 成江涛 Possible mechanisms for transformation of mechanical stimuli to biochemical signals for bone formation Prostaglandin release Electro- magnetic potentials Incr

5、eased bone blood flow Response to microdamage Hormonally mediated mechanisms北京骨外固定技术研究所 市场专员 成江涛 Distraction osteogenesis resembles fracture healing and embryonic bone developmentA series of complex biological cascades of bone repair, regeneration and remodeling unknownTopics of discussion: - Cell p

6、roliferation and death (apoptosis) Cell differentiation Angiogenesis Enhancement of bone formation北京骨外固定技术研究所 市场专员 成江涛 Antrim Coast, Northern IrelandCell Proliferation& Cell Death (Apoptosis)In Fracture Healing北京骨外固定技术研究所 市场专员 成江涛 Study of cell proliferation & cell death in fractureFact: In

7、fracture repair injured bone restores its mechanical integrity and anatomical configuration by original osseous tissue. 北京骨外固定技术研究所 市场专员 成江涛 Hematoma and InflammationAngiogenesisProliferationBone formationBone remodellingApoptosis北京骨外固定技术研究所 市场专员 成江涛 The Mouse Fracture Model (Li, et al, JBMR, No 5,

8、2002)ABCDEDay 2 Day 4 Day 8 Day 16 Day 24北京骨外固定技术研究所 市场专员 成江涛 Day of sacrifice241684Median6005004003002001000Tissue areasBoneCartilageFibrous tissueHistomorphometry北京骨外固定技术研究所 市场专员 成江涛 Cell proliferation by PCNA immunostainingLi, et al, JBMR, 2002CDFGHIABEPPPPPSBPCNA北京骨外固定技术研究所 市场专员 成江涛 CABDEFIHGApo

9、ptosis detected by TUNEL method (Li, et al. JOR, 2003)北京骨外固定技术研究所 市场专员 成江涛 Cell proliferation peaked at day 8 (* p0.05) and declined at day16 - 24. Apoptosis was minimal at day 2-8, peaked at day 16 (* p0.05), and declined at day 24. Li, et al. JBMR, 2002Day of sacrifice241684Pixel ratio600500400300

10、2001000Tissue areasBoneCartilageFibrous tissuePeak of cell proliferationPeak of apoptosisMEAN PCNA AND TUNEL POSITIVELY LABELLED CELLS OF PER HIGH-POWER FIELD 01020304050602481624Days post-fractureMean positive cells per high-power fieldApoptosisCell proliferation北京骨外固定技术研究所 市场专员 成江涛 The current stu

11、dy indicated that cell proliferation and apoptosis are coupled events during fracture repair, cell proliferation is active at the early stages and apoptosis is active during the phase of callus remodelling. 北京骨外固定技术研究所 市场专员 成江涛 Belfast City Hall, Northern Ireland Rate of Distraction&Cell Prolife

12、ration, deathIn Distraction Osteogenesis北京骨外固定技术研究所 市场专员 成江涛 1. What are the effects of distraction rates on bone formation ?2. Is the rate of cell proliferation effected by the rate of distraction ?3. Are the types of tissue formed determined by the rate of distraction ?4. How the unwanted tissues

13、are being removed ?北京骨外固定技术研究所 市场专员 成江涛 Methods Adult male NZW rabbits Unilateral fixator Tibial osteotomy A latency period 7 days Rate of lengthening: 0.3, 0.7 1.3 and 2.7 mm/day Rhythm: 2 increments/day Total lengthening: 20% of the tibia (2 cm)北京骨外固定技术研究所 市场专员 成江涛 sham0.3 mm0.7 mm1.3 mm2.7 mmRadi

14、ography北京骨外固定技术研究所 市场专员 成江涛 0 day -7days- 14 days-21days (stop)- 28daysSerial x-rays of 0.7 mm/day group北京骨外固定技术研究所 市场专员 成江涛 Definition of the zones in the regenerate FZ: Fibrous ZonePMF: Primary Mineralization FrontNBZ: New Bone ZoneLi, et al. Journal of Orthopaedic Research,15:765-72, 1997北京骨外固定技术

15、研究所 市场专员 成江涛 0.3 mm/day1.3 mm/day0.7 mm/day2.7 mm/day北京骨外固定技术研究所 市场专员 成江涛 ABCDResult: Cell ProliferationCell proliferation in the PMF, X 100Cell proliferation in the PMF, X 400Cell proliferation in the NBZ, X 100Cell proliferation in the NBZ, X 400北京骨外固定技术研究所 市场专员 成江涛 Li, et al. J Orthop Res15:765-7

16、2, 1997.The rate of cell proliferation is only affected by the rate of lengthening at the PMF. 北京骨外固定技术研究所 市场专员 成江涛 Question: During distraction osteogenesis new bone is formed and undergoes remodelling rapidly. What are the mechanisms governing the removal of the redundant callus ? Fact: Tissue hom

17、eostasis is achieved by a delicate balance between the cell death (apoptosis) and cell proliferation. 北京骨外固定技术研究所 市场专员 成江涛 ABCDEFTUNEL in PMFTUNEL in FZTUNEL NBZ near PMFNBZ distal to PMFTRAP in NBZTRAPRate: 0.7 mm/day北京骨外固定技术研究所 市场专员 成江涛 ABCEarly apoptosis changes of an OB in the PMFAdvanced apopto

18、sis of an OB in the NBZLater stage of aopotosis of an OB in the NBZBar: 1 mLi, et al, JOR, 2003. 北京骨外固定技术研究所 市场专员 成江涛 Structure of the regenerateExtent of apoptosis in the regenerateExtent of osteoclast activity (TRAP)SummaryCell proliferation and apoptosis co-exist during distraction osteogenesis.T

19、he distribution of apoptotic cells is associated with osteoclast activities. Apoptosis may initiate rapid bone remodeling during distraction osteogenesis. 北京骨外固定技术研究所 市场专员 成江涛 Giants Causeway, N. IrelandCell Differentiation&Distraction Osteogenesis北京骨外固定技术研究所 市场专员 成江涛 Molecular approaches are ba

20、sed on the understanding of bone biology北京骨外固定技术研究所 市场专员 成江涛 北京骨外固定技术研究所 市场专员 成江涛 The expression of BMP-4 is upregulated in the newly formed tissues during distraction osteogenesisLi, et al.Acta Orthop Scand 69:420-425, 1998北京骨外固定技术研究所 市场专员 成江涛 Ossification viachondroid bone Yasui, et alJBJS(Br), 79

21、, 1997Chondrocyte dedifferentiation ?Roach and ScammellJBMR,11, 1996 Cartilage-boneinterface北京骨外固定技术研究所 市场专员 成江涛 Type I collagen in-situ, X100Type II collagen in-situ, X100 Type I, X400Type II, X400北京骨外固定技术研究所 市场专员 成江涛 “ The role of chondrocytes inintramembranous and endochondral ossification during

22、 distraction osteogenesis in the rabbit”Li, et al. Calcif Tissue Int (1999) 64:310-17.HE staining, X100Type II collagen immunostaing, X100Type I collagen in-situ hybridization, X100Chondrocyte - bone cells ? 北京骨外固定技术研究所 市场专员 成江涛 Queens University BelfastCellular metabolism is enhanced in distraction

23、 osteogesis, as well as expressions of genes and proteins associated with bone development. There are shared pathways of fracture healing and distraction osteogenesis, the only principal difference is influence of the mechanical condition. The mechanical stimuli during distraction osteogenesis plays

24、 a key role in tissue regeneration. 北京骨外固定技术研究所 市场专员 成江涛 Belfast CathedralAngiogenesisinFracture&Distraction Osteogenesis北京骨外固定技术研究所 市场专员 成江涛 Fracture北京骨外固定技术研究所 市场专员 成江涛 Angiogenesis during distraction osteogenesis北京骨外固定技术研究所 市场专员 成江涛 Angiographic study: Lead oxide angiography, 0.7 mm/day北京骨外固定

25、技术研究所 市场专员 成江涛 Angiogenesis StudyLi, et al. J Orthop Res17:362-67, 1999.北京骨外固定技术研究所 市场专员 成江涛 Angiogenesis StudyImmunostaining using MAb for type IV collagen1.3 mm/dayNew Bone ZoneX100X400Li, et al. J Orthop Res17:362-67, 1999北京骨外固定技术研究所 市场专员 成江涛 Li, et al. J Orthop Res17:362-67, 1999北京骨外固定技术研究所 市场专员

26、 成江涛 Summary北京骨外固定技术研究所 市场专员 成江涛 Muscuoloskeletal Education and Research UnitQueens University Belfast Enhancement of Bone FormationinDistraction Osteogenesis北京骨外固定技术研究所 市场专员 成江涛 1. Physical stimulation (micro-movement / ultrasound)2. Electronic stimulation (electrode) 3. Surgical intervention (bone

27、 grafting)4. Cell therapy (bone marrow, osteoblast application)4. Molecular intervention (growth factors)5. Genetic approaches (gene therapy) 北京骨外固定技术研究所 市场专员 成江涛 Bone to fill ? Long duration of the bone consolidation phase in DOrhBMP-2 promote bone formation in fracture and segmental bone defect mo

28、dels. (Yasko, et al, 1992; Gerhart, et al. 1993; Kirker-Head, et al., 1995; Welch , et al., 1998; Bax, et al., 1999)Can rhBMP-2 application help ? 北京骨外固定技术研究所 市场专员 成江涛 Methods A latency period 7 days Tibiae lengthened 2 cm over a period of 10 days Rate: 2 mm/day Rhythm: 2 increments/day rhBMP-2 (75

29、g) was administrated to the gap Placebo controls北京骨外固定技术研究所 市场专员 成江涛 Radiographic signs of cortical discontinuity or “crack”Treatment GroupsDays after lengthening14 days28 days(n=4)(n=4)Non-treatment4/43/4ACS / Buffer4/43/4ACS / rhBMP-22/4NoneBuffer / rhBMP-21/4NoneBuffer injection only 4/42/4Li, et

30、 al. rhBMP-2 in Distraction OsteogenesisJournal of Orthopaedic Research, 2002北京骨外固定技术研究所 市场专员 成江涛 北京骨外固定技术研究所 市场专员 成江涛 Non-treatmentBufferinjectionACS/SalineACS/rhBMP-2Buffer/rhBMP-25 days14 days28days14 dNon-treat14dBMP-228 dNon-treat28dBMP-2北京骨外固定技术研究所 市场专员 成江涛 Summary Both injection of rhBMP-2 in

31、 buffer and implantation in ACS significantly enhance the consolidation of the regenerate rhBMP-2/buffer injection may be an appropriate delivery method as it is effective and less invasive than rhBMP-2/ACS implantation. Using rhBMP-2 to enhance distracion osteogenesis may have potential clinical ap

32、plication in reducing complications associated with this procedure.北京骨外固定技术研究所 市场专员 成江涛 Bone Formation During Distraction Osteogenesis is Enhanced by Thrombin Peptide (TP508) 北京骨外固定技术研究所 市场专员 成江涛 TP508 (ChrysalinTM)Synthetically produced fragment of Thrombin23 Amino Acid PeptideCompetes for binding

33、site &stimulates N-PAR signal pathways, without affecting the blood clotting activity of natural thrombin Mimics Thrombin functions in tissue repairTP508 北京骨外固定技术研究所 市场专员 成江涛 Thrombin ReceptorsTwo Thrombin receptorsProteolytically activated receptors (PARs)Non-proteolytically activated receptors

34、 (N-PARs) TP508北京骨外固定技术研究所 市场专员 成江涛 Why Is Thrombin Important in Tissue Repair?Primary EffectsSecondary EffectsCleaves fibrinogen to form clotsActivates blood plateletsIncreases vascular permeability Inflammatory cell adhesionPromote angiogenesisChemotaxis and mitogenesis Proliferation in epithelial

35、 and endothelial cellsProliferation and collagen synthesis in fibroblasts北京骨外固定技术研究所 市场专员 成江涛 X-ray Score: 75% Gap filledGroupMean X-ray ScoresFully united animalsTP508 100 g/kg3.254/8 (50%)TP508 10 g/kg2.712/7 (28.6%)Saline 300 L2.501/6 (16.6%)The radiographic and histology evaluation indicated a s

36、ignificant increase in new bone formation in the distraction regenerates in the TP508 treated groups, compared to the controls post-lengthening. ResultsTP 508100 g/kgTP 50810 g/kgSalineinjectionDay 0 End 1 week 2 week HistologySummary北京骨外固定技术研究所 市场专员 成江涛 Results: HistologyGroup 1: TP508 100g/kgMost of the regenerate is woven bone, with abundant vascularization.Rare fibrous and cartilage tissues北京骨外固定技术研究所 市场专员 成江涛 Results: HistologyGroup 2: TP508 10g/kg Some fibrous and catilage tissues are seen in the regenerate. 60-70% of the regenerate is woven bone.北京骨外固定技术研究所